Low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis

ABSTRACT

Isolated polynucleotides encoding novel polypeptides which are capable of binding to native and methylated LDL (low density lipoprotein), the isolated polypeptides, called LBPs (LDL binding proteins), and biologically active fragments and analogs thereof, are described. Also described are methods for determining if an animal is at risk for atherosclerosis, methods for evaluating an agent for use in treating atherosclerosis, methods for treating atherosclerosis, and methods for treating a cell having an abnormality in structure or metabolism of LBP. Pharmaceutical compositions and vaccine compositions are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/517,849U.S. Pat. No. assigned, filed Mar. 2, 2000, which was a continuation-inpart of U.S. Ser. No. 08/979,608, filed Nov. 26, 1997 now U.S. Pat. No.6,355,451, which claimed priority from U.S. Ser. No. 60/031,930, filedNov. 27, 1996, and now U.S. Ser. No. 60/048,547, filed Jun. 3, 1997.

FIELD OF THE INVENTION

This invention relates to novel polypeptides (LBPs) which bind to lowdensity lipoprotein (LDL), polynucleotides which encode thesepolypeptides, and treatments, diagnoses and therapeutic agents foratherosclerosis.

BACKGROUND OF THE INVENTION

Atherosclerosis is the principal cause of heart attacks and strokes. Ithas been reported that about 50% of all deaths in the United States,Europe and Japan are due to atherosclerosis. Atherosclerotic lesions inthe arterial wall characterize atherosclerosis. Cholesteryl esters (CE)are present in these atherosclerotic lesions. Low density lipoprotein(LDL) has been shown to be the major carrier of plasma CE, and has beenimplicated as the agent by which CE enter the atherosclerotic lesions.

Scattered groups of lipid-filled macrophages, called foam cells, are thefirst visible signs of atherosclerosis and are described as type Ilesions. These macrophages are reported to contain CE derived from LDL.The macrophages recognize oxidized LDL, but not native LDL, and becomefoam cells by phagocytosing oxidized LDL. Larger, more organizedcollections of foam cells, fatty streaks, represent type II lesions.These lesions further develop into complex lesions called plaques, whichcan result in impeding the flow of blood in the artery.

It is widely believed that accumulation of LDL in the artery depends onthe presence of functionally modified endothelial cells in the arterialwall. It has been reported in animal models of atherosclerosis that LDL,both native LDL and methylated LDL, accumulates focally and irreversiblyonly at the edges of regenerating endothelial islands in aortic lesions,where functionally modified endothelial cells are present, but not inthe centers of these islands where endothelial regeneration iscompleted. Similarly, LDL accumulates in human atherosclerotic lesions.The mechanism by which the LDL accumulates focally and irreversibly inarterial lesions has not heretofore been understood.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides which bind toLDL.

It is yet another object of the invention to provide a method fordetermining if an animal is at risk for atherosclerosis.

It is yet another object of the invention to provide a method forevaluating an agent for use in treating atherosclerosis. It is yetanother object of the invention to provide a method for treatingatherosclerosis.

Still another object of the invention is to utilize an LBP (low densitylipoprotein binding protein) gene and/or polypeptide, or fragments,analogs and variants thereof, to aid in the treatment, diagnosis and/oridentification of therapeutic agents for atherosclerosis.

In one aspect, the invention features an isolated polynucleotidecomprising a polynucleotide encoding the polypeptide comprising theamino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8; SEQ ID NO: 9; SEQ ID NO:43; SEQ ID NO:44; SEQ ID NO:47 or apolynucleotide capable of hybridizing to and which is at least about 95%identical to any of the above polynucleotides and wherein the encodedpolypeptide is capable of binding to LDL; or a biologically activefragment of any of the above polynucleotides wherein the encodedpolypeptide is capable of binding to LDL.

In certain embodiments, the polynucleotide comprises the nucleic acidsequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17; SEQ ID NO: 18; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:48.

Another aspect of the invention is an isolated polypeptide comprising apolypeptide having the amino acid sequence as set forth in SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 43; SEQ ID NO:44;SEQ ID NO:47; or a polypeptide which is at least about 95% identical toany of the above polypeptides and wherein the polypeptide is capable ofbinding to LDL; or a biologically active fragment of any of the abovepolypeptides wherein the fragment is capable of binding to LDL.

Another aspect of the invention is a method for determining if an animalis at risk for atherosclerosis. An animal is provided. An aspect of LBPmetabolism or structure is evaluated in the animal. An abnormality inthe aspect of LBP metabolism or structure is diagnostic of being at riskfor atherosclerosis. Another aspect of the invention is a method forevaluating an agent for use in treating atherosclerosis. A test cell,cell-free system or animal is provided. An agent is provided. The agentis administered to the test cell, cell-free system or animal in atherapeutically effective amount. The effect of the agent on an aspectof LBP metabolism or structure is evaluated. A change in the aspect ofLBP metabolism or structure is indicative of the usefulness of the agentin treating atherosclerosis.

Another aspect of the invention is a method for evaluating an agent forthe ability to alter the binding of LBP polypeptide to a bindingmolecule, e.g., native LDL, modified LDL, e.g., methylated LDL oroxidized LDL, or an arterial extracellular matrix structural component.An agent is provided. An LBP polypeptide is provided. A binding moleculeis provided. The agent, LBP polypeptide and binding molecule arecombined. The formation of a complex comprising the LBP polypeptide andbinding molecule is detected. An alteration in the formation of thecomplex in the presence of the agent as compared to in the absence ofthe agent is indicative of the agent altering the binding of the LBPpolypeptide to the binding molecule.

Another aspect of the invention is a method for evaluating an agent forthe ability to bind to an LBP polypeptide. An agent is provided. An LBPpolypeptide is provided. The agent is contacted with the LBPpolypeptide. The ability of the agent to bind to the LBP polypeptide isevaluated.

Another aspect of the invention is a method for evaluating an agent forthe ability to bind to a nucleic acid encoding an LBP regulatorysequence. An agent is provided. A nucleic acid encoding an LBPregulatory sequence is provided. The agent is contacted with the nucleicacid. The ability of the agent to bind to the nucleic acid is evaluated.

Another aspect of the invention is a method for treating atherosclerosisin an animal. An animal in need of treatment for atherosclerosis isprovided. An agent capable of altering an aspect of LBP structure ormetabolism is provided. The agent is administered to the animal in atherapeutically effective amount such that treatment of theatherosclerosis occurs. In certain embodiments, the agent is an LBPpolypeptide, e.g., LBP-1, LBP-2 or LBP-3, or a biologically activefragment or analog thereof. In certain embodiments, the agent is apolypeptide of no more than about 100, 50, 30, 20, 10, 5, 4, 3 or 2amino acid residues in length. In certain embodiments, the agent is apolypeptide having an amino acid sequence that includes at least about20%, 40%, 60%, 80%, 90%, 95% or 98% acidic amino acid residues.

Another aspect of the invention is a method for treating an animal atrisk for atherosclerosis. An animal at risk for atherosclerosis isprovided. An agent capable of altering an aspect of LBP structure ormetabolism is provided. The agent is administered to the animal in atherapeutically effective amount such that treatment of the animaloccurs.

Another aspect of the invention is a method for treating a cell havingan abnormality in structure or metabolism of LBP. A cell having anabnormality in structure or metabolism of LBP is provided. An agentcapable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the cell in a therapeuticallyeffective amount such that treatment of the cell occurs.

Another aspect of the invention is a pharmaceutical composition fortreating atherosclerosis in an animal comprising a therapeuticallyeffective amount of an agent, the agent being capable of altering anaspect of LBP metabolism or structure in the animal so as to result intreatment of the atherosclerosis, and a pharmaceutically acceptablecarrier.

Another aspect of the invention is a vaccine composition for treatingatherosclerosis in an animal comprising a therapeutically effectiveamount of an agent, the agent being capable of altering an aspect of LBPmetabolism or structure in the animal so as to result in treatment ofthe atherosclerosis, and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for diagnosingatherosclerotic lesions in an animal. An animal is provided. A labeledagent capable of binding to LBP, e.g., LBP-1, LBP-2 or LBP-3, present inatherosclerotic lesions is provided. The labeled agent is administeredto the animal under conditions which allow the labeled agent to interactwith the LBP so as to result in labeled LBP. The localization orquantification of the labeled LBP is determined by imaging so as todiagnose the presence of atherosclerotic lesions in the animal.

Another aspect of the invention is a method for immunizing an animalagainst an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or analogthereof. An animal having LDL is provided. The LBP or fragment or analogthereof is administered to the animal so as to stimulate antibodyproduction by the animal to the LBP or fragment or analog thereof suchthat binding of the LBP to the LDL is altered, e.g., decreased orincreased.

Another aspect of the invention is a method of making a fragment oranalog of LBP polypeptide, the fragment or analog having the ability tobind to native LDL and to modified LDL, e.g., methylated LDL, oxidizedLDL, acetylated LDL, or cyclohexanedione-treated LDL. An LBP polypeptideis provided. The sequence of the LBP polypeptide is altered. The alteredLBP polypeptide is tested for the ability to bind to modified LDL andnative LDL.

Yet another aspect of the invention is a method for isolating a cDNAencoding an LBP. A cDNA library is provided. The cDNA library isscreened for a cDNA encoding a polypeptide which binds to native LDL andmodified LDL, e.g., methylated LDL or oxidized LDL. The cDNA whichencodes the polypeptide is isolated, the cDNA encoding an LBP.

The above and other features, objects and advantages of the presentinvention will be better understood by a reading of the followingspecification in conjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of rabbit LBP-1 (SEQ ID NO: 1).Differences in amino acids between rabbit and human LBP-1 are depictedin bold type.

FIGS. 2A-1 to 2A-3 depict the nucleotide sequence (SEQ ID NO: 48) andamino acid sequence (SEQ ID NO: 47) of rabbit LBP-2.

FIG. 2B depicts a portion of the amino acid sequence of rabbit LBP-2(SEQ ID NO: 2). Differences in amino acids between rabbit and humanLBP-2 are depicted in bold type. Where the sequences depicted in FIG. 2Aand FIG. 2B differ, FIG. 2A represents the rabbit LBP-2 sequence.

FIG. 3 depicts the amino acid sequence of amino acids 319 to 550 ofrabbit LBP-2 (SEQ ID NO: 3).

FIG. 4 depicts the amino-acid sequence of amino acids 299 to 550 ofrabbit LBP-2 (SEQ ID No: 4).

FIGS. 5A to 5B depict the amino acid sequence of rabbit LBP-3 (SEQ IDNO: 5). Differences in amino acids between rabbit and human LBP-3 aredepicted in bold type.

FIG. 6 depicts the amino acid sequence of human LBP-1 (SEQ ID NO: 6).Differences in amino acids between rabbit and human LBP-1 are depictedin bold type.

FIGS. 7A-1 to 7A-3 depict the nucleotide sequence (SEQ ID NO: 45) andamino acid sequence (SEQ ID NO: 43) of human LBP-2.

FIG. 7B depicts the amino acid sequence of amino acids 322 to 538 ofhuman LBP-2 (SEQ ID NO: 7). Differences in amino acids between rabbitand human LBP-2 are depicted in bold type.

FIGS. 8A-1 to 8A-3 depict the nucleotide sequence (SEQ ID NO: 46) andamino acid sequence (SEQ ID NO: 44) of human LBP-3.

FIGS. 8B-1 to 8B-2 depict the amino acid sequence of amino acids 17 to546 of human LBP-3 (SEQ ID NO: 8). Differences in amino acids betweenrabbit and human LBP-3 are depicted in bold type. Where the sequencesdepicted in FIG. 8A and FIG. 8B differ, FIG. 8A represents the humanLBP-3 sequence.

FIG. 9 depicts the amino acid sequence of amino acids 14 to 33 of humanor rabbit LBP-1, called BHF-1 (SEQ ID NO: 9).

FIGS. 10A to 10B depict the cDNA sequence encoding rabbit LBP-1 (SEQ IDNO: 10) and the corresponding amino acid sequence (SEQ ID NO:1).Differences in amino acids between rabbit and human LBP-1 are depictedin bold type.

FIGS. 11A to 11C depict a cDNA sequence encoding a portion of rabbitLBP-2 (SEQ ID NO: 11) and the corresponding amino acid sequence (SEQ IDNO:2). Differences in amino acids between rabbit and human LBP-2 aredepicted in bold type. Where the sequences depicted in FIG. 2A and FIG.11 differ, FIG. 2A represents the rabbit LBP-2 sequence.

FIGS. 12A to 12B depict a cDNA sequence of nucleotides 256 to 1617 (SEQID NO: 12) of SEQ ID NO: 11 of rabbit LBP-2 and the corresponding aminoacid sequence (SEQ ID NO:3).

FIGS. 13A to 13B depict a cDNA sequence of nucleotides 196 to 1617 (SEQID NO: 13) of SEQ ID NO: 11 of rabbit LBP-2 and the corresponding aminoacid sequence (SEQ ID NO:4).

FIGS. 14A to 14F depict the cDNA sequence encoding rabbit LBP-3 (SEQ IDNO: 14) and the corresponding amino acid sequence (SEQ ID NO:5).Differences in amino acids between rabbit and human LBP-3 are depictedin bold type.

FIGS. 15A to 15B depict the cDNA sequence encoding human LBP-1 (SEQ IDNO: 15) and the Add corresponding amino acid sequence (SEQ ID NO:6).Differences in amino acids between rabbit and human LBP-1 are depictedin bold type.

FIGS. 16A to 16B depict a cDNA sequence encoding a portion of humanLBP-2 (SEQ ID NO: 16) and the corresponding amino acid sequence (SEQ IDNO:7). Differences in amino acids between rabbit and human LBP-2 aredepicted in bold type.

FIGS. 17A to 17D depict a cDNA sequence encoding a portion of humanLBP-3 (SEQ ID NO: 17) and the corresponding amino acid sequence (SEQ IDNO:8). Differences in amino acids between rabbit and human LBP-3 aredepicted in bold type. Where the sequences depicted in FIG. 8A and FIG.17 differ, FIG. 8A represents the human LBP-3 sequence.

FIG. 18 depicts the cDNA sequence encoding BHF-1 (SEQ ID NO: 18) andcorresponding amino acid sequence (SEQ ID NO:9).

FIG. 19 corresponds to the amino acid sequence of rabbit LBP-1 (topsequence; SEQ ID NO:1) in alignment with the amino acid sequence ofhuman LBP-1 (bottom sequence; SEQ ID NO:6).

FIG. 20 corresponds to the amino acid sequence of a portion of the aminoacid sequence of rabbit LBP-2 (top sequence; amino acid residues 331-550of SEQ ID NO:47) in alignment with a portion of the amino acid sequenceof human LBP-2 (bottom sequence; SEQ ID NO:7).

FIG. 21 corresponds to the amino acid sequence of rabbit LBP-3 (topsequence; SEQ ID NO:5) in alignment with the amino acid sequence of aportion of human LBP-3 (bottom sequence; SEQ ID NO:44).

FIGS. 22A to 22E depict the genomic sequence of human LBP-1 (SEQ IDNO:49) and corresponding amino acid sequence (SEQ ID NO:6).

FIGS. 23A to 23F depict the genomic sequence of human LBP-2 (SEQ IDNO:50) and corresponding amino acid sequence (SEQ ID NO:43).

FIGS. 24A to 24I depict the genomic sequence of human LBP-3 (SEQ IDNO:51) and corresponding amino acid sequence (SEQ ID NO:44).

DETAILED DESCRIPTION

In accordance with aspects of the present invention, there are providednovel mature human and rabbit polypeptides, LBP-1, LBP-2 and LBP-3, andbiologically active analogs and fragments thereof, and there areprovided isolated polynucleotides which encode such polypeptides. LBP isan abbreviation for low density lipoprotein (LDL) binding protein. Theterms polynucleotide, nucleotide and oligonucleotide are usedinterchangeably herein, and the terms polypeptides, proteins andpeptides are used interchangeably herein.

This invention provides for an isolated polynucleotide comprising apolynucleotide encoding the polypeptide having the amino acid sequenceof rabbit LBP-1 as set forth in FIG. 1 (SEQ ID NO: 1); rabbit LBP-2 asset forth in FIG. 2A (SEQ ID NO: 47); a portion of rabbit LBP-2 as setforth in FIG. 2B (SEQ ID NO: 2); 319 to 550 of rabbit LBP-2 as set forthin FIG. 3 (SEQ ID NO: 3); 299 to 550 of rabbit LBP-2 as set forth inFIG. 4 (SEQ ID NO: 4); rabbit LBP-3 as set forth in FIG. 5 (SEQ ID NO:5); human LBP-1 as set forth in FIG. 6 (SEQ ID NO: 6); human LBP-2 asset forth in FIG. 7A (SEQ ID NO: 43); 322 to 538 of human LBP-2 as setforth in FIG. 7B (SEQ ID NO: 7); human LBP-3 as set forth in FIG. 8A(SEQ ID NO: 44); 17-546 of human LBP-3 as set forth in FIG. 8B (SEQ IDNO: 8); 14 to 33 of human (SEQ ID NO:6) or rabbit (SEQ ID NO: 1) LBP-1,called BHF-1, as set forth in FIG. 9 (SEQ ID NO: 9); a polynucleotidecapable of hybridizing to and which is at least about 80% identical,more preferably at least about 90% identical, more preferably yet atleast about 95% identical, and most preferably at least about 98%identical to any of the above polynucleotides, and wherein the encodedpolypeptide is capable of binding to LDL; or a biologically activefragment of any of the above polynucleotides wherein the encodedpolypeptide is capable of binding to LDL.

This invention also includes an isolated polynucleotide comprising apolynucleotide encoding the polypeptide having amino acid residues329-343 (SEQ ID NO: 19), 329-354 (SEQ ID NO: 20), 344-354 (SEQ ID NO:21) or 529-538 (SEQ ID NO: 22) of human LBP-2 as set forth in FIG. 7A(SEQ ID NO: 43); amino acid residues 14-43 (SEQ ID NO: 23) or 38-43 (SEQID NO: 24) of rabbit or human LBP-1 as set forth in FIG. 1 (SEQ IDNO: 1) and FIG. 6 (SEQ ID NO: 6); amino acid residues 338-353 (SEQ IDNO: 25), 338-365 (SEQ ID NO: 26), 354-365 (SEQ ID NO: 27) or 444-453(SEQ ID NO: 28) of rabbit LBP-2 as set forth in FIG. 2A (SEQ ID NO: 47);amino acid residues 96-110 (SEQ ID NO: 29) of rabbit LBP-3 as set forthin FIG. 5 (SEQ ID NO: 5); amino acid residues 69-75 (SEQ ID NO: 41) ofhuman LBP-3 as set forth in FIG. 8A (SEQ ID NO: 44); a polynucleotidecapable of hybridizing to and which is at least about 80% identical,more preferably at least about 90% identical, more preferably yet atleast about 95% identical, and most preferably at least about 98%identical to any of the above polynucleotides, and wherein the encodedpolypeptide is capable of binding to LDL; or a biologically activefragment of any of the above polynucleotides wherein the encodedpolypeptide is capable of binding to LDL.

By a polynucleotide encoding a polypeptide is meant a polynucleotidewhich includes only coding sequence for the polypeptide, as well as apolynucleotide which includes additional coding and/or non-codingsequences. Thus, e.g., the polynucleotides which encode for the maturepolypeptides of FIGS. 1-9 (SEQ ID NOS: 1-9, 43, 44 and 47) may includeonly the coding sequence for the mature polypeptide; the coding sequencefor the mature polypeptide and additional coding sequence such as aleader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequences 5′ and/or 3′ of the coding sequence for the maturepolypeptide. The polynucleotides of the invention are also meant toinclude polynucleotides in which the coding sequence for the maturepolypeptide is fused in the same reading frame to a polynucleotidesequence which aids in expression and/or secretion of a polypeptide froma host cell, e.g., a leader sequence. The polynucleotides are also meantto include polynucleotides in which the coding sequence is fused inframe to a marker sequence which, e.g., allows for purification of thepolypeptide.

The polynucleotides of the present invention may be in the form of RNA,DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or synthetic DNA, RNA or PNA.The DNA may be double-stranded or single stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand.

In preferred embodiments, the polynucleotide comprises the nucleic acidof rabbit LBP-1 as set forth in FIG. 10 (SEQ ID NO: 10); rabbit LBP-2 asset forth in FIG. 2A (SEQ ID NO:48) or FIG. 11 (SEQ ID NO:11);nucleotide 256 to 1617 of SEQ ID NO: 11 of rabbit LBP-2 as set forth inFIG. 12 (SEQ ID NO: 12); nucleotide 196 to 1617 of SEQ ID NO: 11 ofrabbit LBP-2 as set forth in FIG. 13 (SEQ ID NO: 13); rabbit LBP-3 asset forth in FIG. 14 (SEQ ID NO: 14); human LBP-1 as set forth in FIG.15 (SEQ ID NO: 15); human LBP-2 as set forth in FIG. 7A (SEQ ID NO: 45)or FIG. 16 (SEQ ID NO: 16); human LBP-3 as set forth in FIG. 8A (SEQ IDNO: 46) or FIG. 17 (SEQ ID NO: 17); or nucleotide 97 to 156 of rabbitLBP-1 or nucleotide 157 to 216 of human LBP-1, (BHF-1), as set forth inFIG. 18 (SEQ ID NO: 18).

In other preferred embodiments, the polynucleotide comprises the nucleicacid as set forth in SEQ ID NO:30(GAAGAGGAAGAAGATGATGATGAAGATGAAGATGAAGAAGATGAT), SEQ ID NO:31(GAAGAGGAAGAAGATGATGATGAAGATGAAGATGAAGAAGA TGATGTGTCAGAGGGCTCTGAAGTGCCCGAGAGTGAC), SEQ ID NO:32(GTGTCAGAGGGCTCTGAAGTGCCCGAGAGTGAC), SEQ ID NO:33(GAGGATGATGACCCCGATGGCTTCTTAGGC), SEQ ID NO:34(GTGGACGTGGATGAATATGACGAGAACAAGTTCGTGGACGAAGAAGATGGGGGCGACGGCCAGGCCGGGCCCGACGAGGGCGAGGTGGAC), SEQ ID NO:35(GACGAGGGCGAGGTGGAC), SEQ ID NO:36(GAGGAGGAGGAGGAGGAGGAGGAAGACGACGAGGACGACG ACGACGAC), SEQ ID NO:37(GAGGAGGAGGAGGAGGAGGAGGAAGACGACGAGGACGACGACGACGACGTCGTGTCCGAGGGCTCGGAGGTGCCCGAGAGCGAT), SEQ ID NO:38(GTCGTGTCCGAGGGCTCGGAGGTGCCCGAGAGCGAT), SEQ ID NO:39(CCCCCCGGGAAGCCAGCCCTCCCAGGAGCC), SEQ ID NO:40(GAGGATGGGGTCCAGGGTGAGCCCCCTGAACCTGAAGATGCA GAG), or SEQ ID NO:42(CGTGATGTCTCTGAGGAGCTG).

The coding sequence which encodes the mature polypeptide may beidentical to the coding sequences shown in FIGS. 2A, 7A, 8A and 10-18(SEQ ID NOS: 10-18, 45, 46, and 48) or SEQ ID NOS: 30-40 or 42, or maybe a different coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same maturepolypeptides as the DNA of FIGS. 2A, 7A, 8A and 10-18 (SEQ ID NOS:10-18, 45, 46, and 48) and SEQ ID NOS: 30-40 and 42.

This invention also includes recombinant vectors comprising thepolynucleotides described above. The vector can be, e.g., a plasmid, aviral particle or a phage. In certain embodiments, the recombinantvector is an expression vector. The vectors may also include variousmarker genes which are useful in identifying cells containing suchvectors.

This invention also includes a cell comprising such a recombinantvector. The recombinant vectors described herein can be introduced intoa host cell, e.g., by transformation, transfection or infection.

This invention also includes a method for producing an LBP comprisingculturing such a cell under conditions that permit expression of theLBP.

This invention also includes an isolated polypeptide comprising apolypeptide having the amino acid sequence as set forth in FIG. 1 (SEQID NO: 1); FIG. 2A (SEQ ID NO: 47); FIG. 2B (SEQ ID NO: 2); FIG. 3 (SEQID NO: 3); FIG. 4 (SEQ ID NO: 4); FIG. 5 (SEQ ID NO: 5); FIG. 6 (SEQ IDNO: 6); FIG. 7A (SEQ ID NO: 43); FIG. 7B (SEQ ID NO: 7); FIG. 8A (SEQ IDNO: 44); FIG. 8B (SEQ ID NO: 8); or FIG. 9 (SEQ ID NO: 9); or apolypeptide which is at least about 80% identical, more preferably atleast about 90% identical, more preferably yet at least about 95%identical, and most preferably at least about 98% identical to the abovepolypeptides, and wherein said polypeptide is capable of binding to LDL;or a biologically active fragment of any of the above polypeptideswherein the fragment is capable of binding to LDL. Differences in aminoacids between the rabbit and human LBP-1, LBP-2 and LBP-3 genes aredepicted in bold type in the FIGURES. Differences in the amino acidsequences between rabbit and human LBP-1, LBP-2 and LBP-3 are alsospecifically shown in FIGS. 19, 20 and 21, respectively.

This invention also includes an isolated polypeptide comprising apolypeptide having amino acid residues 329-343 (SEQ ID NO: 19), 329-354(SEQ ID NO: 20), 344-354 (SEQ ID NO: 21) or 529-538 (SEQ ID NO: 22) asset forth in FIG. 7A (SEQ ID NO: 47); amino acid residues 14-43 (SEQ IDNO: 23) or 38-43 (SEQ ID NO: 24) as set forth in FIG. 1 (SEQ ID NO: 1)and FIG. 6 (SEQ ID NO: 6); amino acid residues 338-353 (SEQ ID NO: 25),338-365 (SEQ ID NO: 26), 354-365 (SEQ ID NO: 27) or 444-453 (SEQ ID NO:28) as set forth in FIG. 2A (SEQ ID NO: 47); amino acid residues 96-110(SEQ ID NO: 29) as set forth in FIG. 5 (SEQ ID NO: 5); and amino acidresidues 69-75 (SEQ ID NO: 41) as set forth in FIG. 8A (SEQ ID NO:44);or a polypeptide which is at least about 80% identical, more preferablyat least about 90% identical, more preferably yet at least about 95%identical, and most preferably at least about 98% identical to the abovepolypeptides, and wherein said polypeptide is capable of binding to LDL;or a biologically active fragment of any of the above polypeptideswherein the fragment is capable of binding to LDL.

The polypeptides of the invention are meant to include, e.g., anaturally purified product, a chemically synthesized product, and arecombinantly derived product.

The polypeptides can be used, e.g., to bind to LDL, thereby inhibitingformation of atherosclerotic plaques. The polypeptides can also be used,e.g., in gene therapy, by expression of such polypeptides in vivo. Thepolypeptides can also be used in pharmaceutical or vaccine compositions.The polypeptides can also be used as immunogens to produce antibodiesthereto, which in turn, can be used as antagonists to the LBPpolypeptides.

Without being bound by any theory, it is believed that the LBPs providethe mechanism by which atherosclerosis is promoted through LDLoxidation. The LBPs are believed to be required in order for focal,irreversible LDL binding to occur at the arterial wall, and that suchbinding is a critical early event in atherosclerosis because it allowsthe time necessary for LDL to be changed from its native state to afully oxidized state.

Since oxidized, but not native, LDL is a foreign protein, macrophagesingest it, first becoming the foam cells of type I lesions, andsubsequently forming the fatty streaks of type II lesions.

This invention also includes a method for determining if an animal is atrisk for atherosclerosis. An animal is provided. An aspect of LBPmetabolism or structure is evaluated in the animal. An abnormality inthe aspect of LBP metabolism or structure is diagnostic of being at riskfor atherosclerosis.

By atherosclerosis is meant a disease or condition which comprisesseveral stages which blend imperceptibly into each other, includingirreversible binding of LDL, LDL oxidation, macrophage recruitment,blockage of the artery and tissue death (infarction).

By animal is meant human as well as non-human animals. Nonhuman animalsinclude, e.g., mammals, birds, reptiles, amphibians, fish, insects andprotozoa. Preferably, the nonhuman animal is a mammal, e.g., a rabbit, arodent, e.g., a mouse, rat or guinea pig, a primate, e.g., a monkey, ora pig. An animal also includes transgenic non-human animals. The termtransgenic animal is meant to include an animal that has gained newgenetic information from the introduction of foreign DNA, i.e., partlyor entirely heterologous DNA, into the DNA of its cells; or introductionof a lesion, e.g., an, in vitro induced mutation, e.g., a deletion orother chromosomal rearrangement into the DNA of its cells; orintroduction of homologous DNA into the DNA of its cells in such a wayas to alter the genome of the cell into which the DNA is inserted, e.g.,it is inserted at a location which differs from that of the natural geneor its insertion results in a knockout or replacement of the homologoushost gene or results in altered and/or regulatable expression and/ormetabolism of the gene. The animal may include a transgene in all of itscells including germ line cells, or in only one or some of its cells.Transgenic animals of the invention can serve as a model for studyingatherosclerosis or for evaluating agents to treat atherosclerosis.

In certain embodiments, the determination for being at risk foratherosclerosis is done in a prenatal animal.

By LBP is meant a low density lipoprotein (LDL) binding protein which iscapable of binding LDL and methylated LDL. By methylated LDL is meantthat about 50% to about 90% of the lysine residues of LDL have a methylgroup chemically attached. Methylated LDL is not recognized bypreviously reported cell surface receptors. See, e.g., Weisgraber etal., J. Biol. Chem. 253: 9053-9062 (1978). In certain embodiments, theLBP is also capable of binding oxidized LDL. In certain preferredembodiments, the binding of LDL to an LBP is irreversible. In certainpreferred embodiments, the LBP does not transport the LDL to anyintracellular compartment. Examples of LBPs are LBP-1, LBP-2 and LBP-3described herein.

By LBP metabolism is meant any aspect of the production, release,expression, function, action, interaction or regulation of LBP. Themetabolism of LBP includes modifications, e.g., covalent or non-covalentmodifications, of LBP polypeptide. The metabolism of LBP includesmodifications, e.g., covalent or noncovalent modifications, that LBPinduces in other substances. The metabolism of LBP also includes changesin the distribution of LBP polypeptide, and changes LBP induces in thedistribution of other substances.

Any aspect of LBP metabolism can be evaluated. The methods used arestandard techniques known to those skilled in the art and can be foundin standard references, e.g., Auaubel et al., ed., Current Protocols inMol. Biology, New York: John Wiley & Sons, 1990; Kriegler, M., ed., GeneTransfer and Expression, Stockton Press, New York, N.Y., 1989; pDisplaygene expression system (Invitrogen, Carlsbad, CA). Preferred examples ofLBP metabolism that can be evaluated include the binding activity of LBPpolypeptide to a binding molecule, e.g., LDL; the transactivationactivity of LBP polypeptide on a target gene; the level of LBP protein;the level of LBP mRNA; the level of LBP modifications, e.g.,phosphorylation, glycosylation or acylation; or the effect of LBPexpression on transfected mammalian cell binding of LDL.

By binding molecule is meant any molecule to which LBP can bind, e.g., anucleic acid, e.g., a DNA regulatory region, a protein, e.g., LDL, ametabolite, a peptide mimetic, a non-peptide mimetic, an antibody, orany other type of ligand. In certain preferred embodiments, the aspectof LBP metabolism that is evaluated is the ability of LBP to bind tonative LDL and/or methylated LDL and/or oxidized LDL. Binding to LDL canbe shown, e.g., by antibodies against LDL, affinity chromatography,affinity coelectrophoresis (ACE) assays, or ELISA assays. See Examples.In other embodiments, it is the ability of LBP to bind to an arterialextracellular matrix structural component that is evaluated. Examples ofsuch components include proteoglycans, e.g., chondroitin sulfateproteoglycans and heparin sulfate proteoglycans; elastin; collagen;fibronectin; vitronectin; integrins; and related extracellular matrixmolecules. Binding to arterial extracellular matrix structuralcomponents can be shown by standard methods known to those skilled inthe art, e.g., by ELISA assays. Primary antibodies to the LBP are thenadded, followed by an enzyme-conjugated secondary antibody to theprimary antibody, which produces a stable color in the presence of anappropriate substrate, and color development on the plates is measuredin a microtiter plate reader.

Transactivation of a target gene by LBP can be determined, e.g., in atransient transfection assay in which the promoter of the target gene islinked to a reporter gene, e.g., β-galactosidase or luciferase, andco-transfected with an LBP expression vector. Such evaluations can bedone in vitro or in vivo. Levels of LBP protein, mRNA orphosphorylation, can be measured, e.g., in a sample, e.g., a tissuesample, e.g., arterial wall, by standard methods known to those skilledin the art.

In certain embodiments, an aspect of LBP structure is evaluated, e.g.,LBP gene structure or LBP protein structure. For example, primary,secondary or tertiary structures can be evaluated. For example, the DNAsequence of the gene is determined and/or the amino acid sequence of theprotein is determined. Standard cloning and sequencing methods can beused as are known to those skilled in the art. In certain embodiments,the binding activity of an antisense nucleic acid with the cellular LBPmRNA and/or genomic DNA is determined using standard methods known tothose skilled in the art so as to detect the presence or absence of thetarget mRNA or DNA sequences to which the antisense nucleic acid wouldnormally specifically bind.

The risk for atherosclerosis that is determined can be a reduced risk oran increased risk as compared to a normal animal. For example, anabnormality which would give a reduced risk is an inactive LBPpolypeptide. An abnormality which would give an increased risk would be,e.g., an LBP polypeptide that has higher activity, e.g., LDL bindingactivity, than native LBP polypeptide.

The invention also includes a method for evaluating an agent for use intreating atherosclerosis. A test cell, cell-free system or animal isprovided. An agent is provided. The agent is administered to the testcell, cell-free system or animal in a therapeutically effective amount.The effect of the agent on an aspect of LBP metabolism or structure isevaluated. A change in the aspect of LBP metabolism or structure isindicative of the usefulness of the agent in treating atherosclerosis.

In certain embodiments, the method employs two phases for evaluating anagent for use in treating atherosclerosis, an initial in vitro phase andthen an in vivo phase. The agent is administered to the test cell orcell-free system in vitro, and if a change in an aspect of LBPmetabolism occurs, then the agent is further administered to a testanimal in a therapeutically effective amount and evaluated in vivo foran effect of the agent on an aspect of LBP metabolism.

By cell is meant a cell or a group of cells, or a cell that is part ofan animal. The cell can be a human or non-human cell. Cell is also meantto include a transgenic cell. The cell can be obtained, e.g., from aculture or from an animal. Animals are meant to include, e.g., naturalanimals and non-human transgenic animals. In certain embodiments, thetransgenic cell or nonhuman transgenic animal has an LBP transgene, orfragment or analog thereof. In certain embodiments, the transgenic cellor non-human transgenic animal has a knockout for the LBP gene.

The test cell, cell-free system or animal can have a wild type patternor a non-wild type pattern of LBP metabolism. A non-wild type pattern ofLBP metabolism can result, e.g., from under-expression, over-expression,no expression, or a temporal, site or distribution change. Such anon-wild type pattern can result, e.g., from one or more mutations inthe LBP gene, in a binding molecule gene, a regulatory gene, or in anyother gene which directly or indirectly affects LBP metabolism. Amutation is meant to include, e.g., an alteration, e.g., in gross orfine structure, in a nucleic acid. Examples include single base pairalterations, e.g., missense or nonsense mutations, frameshifts,deletions, insertions and translocations. Mutations can be dominant orrecessive. Mutations can be homozygous or heterozygous. Preferably, anaspect of LBP-1, LBP-2 or LBP-3 metabolism is evaluated.

An agent is meant to include, e.g., any substance, e.g., ananti-atherosclerosis drug. The agent of this invention preferably canchange an aspect of LBP metabolism. Such change can be the result of anyof a variety of events, including, e.g., preventing or reducinginteraction between LBP and a binding molecule, e.g., LDL or an arterialextracellular matrix structural component; inactivating LBP and/or thebinding molecule, e.g., by cleavage or other modification; altering theaffinity of LBP and the binding molecule for each other; diluting outLBP and/or the binding molecule; preventing expression of LBP and/or thebinding molecule; reducing synthesis of LBP and/or the binding molecule;synthesizing an abnormal LBP and/or binding molecule; synthesizing analternatively spliced LBP and/or binding molecule; preventing orreducing proper conformational folding of LBP and/or the bindingmolecule; modulating the binding properties of LBP and/or the bindingmolecule; interfering with signals that are required to activate ordeactivate LBP and/or the binding molecule; activating or deactivatingLBP and/or the binding molecule in such a way as to prevent binding; orinterfering with other receptors, ligands or other molecules which arerequired for the normal synthesis or functioning of LBP and/or thebinding molecule. For example, the agent can block the binding site onLDL for LBPs expressed focally in the arterial wall extracellularmatrix, or it could block the binding site on an LBP for LDL, or itcould be bifunctional, i.e., it could block both binding sites.

Examples of agents include LBP polypeptide, e.g., LBP-1, LBP-2 or LBP-3,or a biologically active fragment or analog thereof; a nucleic acidencoding LBP polypeptide or a biologically active fragment or analogthereof; a nucleic acid encoding an LBP regulatory sequence or abiologically active fragment or analog thereof; a binding molecule forLBP polypeptide; a binding molecule for LBP nucleic acid, the LBPnucleic acid being, e.g., a nucleic acid comprising a regulatory regionfor LBP or a nucleic acid comprising a structural region for LBP or abiologically active fragment of LBP; an antisense nucleic acid; amimetic of LBP or a binding molecule; an antibody for LBP or a bindingmolecule; a metabolite; or an inhibitory carbohydrate or glycoprotein.In certain embodiments, the agent is an antagonist, agonist or superagonist.

Knowledge of the existence of the sequence of the LBPs allows a searchfor natural or artificial ligands to regulate LDL levels in thetreatment of atherosclerosis. In certain embodiments, the agent is anatural ligand for LBP. In certain embodiments, the agent is anartificial ligand for LBP.

By analog is meant a compound that differs from naturally occurring LBPin amino acid sequence or in ways that do not involve sequence, or both.Analogs of the invention generally exhibit at least about 80% homology,preferably at least about 90% homology, more preferably yet at leastabout 95% homology, and most preferably at least about 98% homology,with substantially the entire sequence of a naturally occurring LBPsequence, preferably with a segment of about 100 amino acid residues,more preferably with a segment of about 50 amino acid residues, morepreferably yet with a segment of about 30 amino acid residues, morepreferably yet with a segment of about 20 amino acid residues, morepreferably yet with a segment of about 10 amino acid residues, morepreferably yet with a segment of about 5 amino acid residues, morepreferably yet with a segment of about 4 amino acid residues, morepreferably yet with a segment of about 3 amino acid residues, and mostpreferably with a segment of about 2 amino acid residues. Non-sequencemodifications include, e.g., in vivo or in vitro chemicalderivatizations of LBP. Non-sequence modifications include, e.g.,changes in phosphorylation, acetylation, methylation, carboxylation, orglycosylation. Methods for making such modifications are known to thoseskilled in the art. For example, phosphorylation can be modified byexposing LBP to phosphorylation-altering enzymes, e.g., kinases orphosphatases. Preferred analogs include LBP or biologically activefragments thereof whose sequences differ from the wild-type sequence byone or more conservative amino acid substitutions or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish LBP biological activity. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. Other examples ofconservative substitutions are shown in Table 1.

TABLE 1 CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid CodeReplace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met,D-Ile, Orn, D-Orn, L- NMMA, L-NAME Asparagine N D-Asn, Asp, D-Asp, Glu,D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln,D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine QD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala AcpHistidine H D-His Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp,Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline PD-Pro, L-I-thioazolidine-4-carboxylic acid, D- orL-1-oxazolidine-4-carboxlic acid Serine S D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tryptophan WD-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Amino acid sequence variants of a protein can be prepared by any of avariety of methods known to those skilled in the art. For example,random mutagenesis of DNA which encodes a protein or a particular domainor region of a protein can be used, e.g., PCR mutagenesis (using, e.g.,reduced Taq polymerase fidelity to introduce random mutations into acloned fragment of DNA; Leung et al., BioTechnique 1: 11-15 (1989)), orsaturation mutagenesis (by, e.g., chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complementary DNAstrand; Mayers et al., Science 229: 242 (1985)). Random mutagenesis canalso be accomplished by, e.g., degenerate oligonucleotide generation(using, e.g., an automatic DNA synthesizer to chemically synthesizedegenerate sequences; Narang, Tetrahedron 39: 3 (1983); Itakura et al.,Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A. G.Walton, Amsterdam: Elsevier, pp. 273-289 (1981)). Non-random or directedmutagenesis can be used to provide specific sequences or mutations inspecific regions. These techniques can be used to create variants whichinclude, e.g., deletions, insertions, or substitutions, of residues ofthe known amino acid sequence of a protein. The sites for mutation canbe modified individually or in series, e.g., by (i) substituting firstwith conserved amino acids and then with more radical choices dependingupon results achieved, (ii) deleting the target residue, (iii) insertingresidues of the same or a different class adjacent to the located site,or (iv) combinations of the above. For example, analogs can be made byin vitro DNA sequence modifications of the sequences of FIGS. 2A, 7A,8A, 10-18 (SEQ ID NOS: 10-18, 45, 46, and 48). For example, in vitromutagenesis can be used to convert any of these DNA sequences into asequence which encodes an analog in which one or more amino acidresidues has undergone a replacement, e.g., a conservative replacementas described in Table 1.

Methods for identifying desirable mutations include, e.g., alaninescanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085(1989)), oligonucleotide-mediated mutagenesis (Adelman et al., DNA, 2:183 (1983)); cassette mutagenesis (Wells et al., Gene 34: 315 (1985)),combinatorial mutagenesis, and phage display libraries (Ladner et al.,PCT International Appln. No. WO88/06630). The LBP analogs can be tested,e.g., for their ability to bind to LDL and/or to an arterialextracellular matrix component, as described herein. Other analogswithin the invention include, e.g., those with modifications whichincrease peptide stability. Such analogs may contain, e.g., one or morenon-peptide bonds (which replace the peptide bonds) in the peptidesequence. Also included are, e.g.: analogs that include residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornonnaturally occurring or synthetic amino acids, e.g., β or γ aminoacids; and cyclic analogs.

Analogs are also meant to include peptides in which structuralmodifications have been introduced into the peptide backbone so as tomake the peptide non-hydrolyzable. Such peptides are particularly usefulfor oral administration, as they are not digested. Peptide backbonemodifications include, e.g., modifications of the amide nitrogen, theα-carbon, the amide carbonyl, or the amide bond, and modificationsinvolving extensions, deletions or backbone crosslinks. For example, thebackbone can be modified by substitution of a sulfoxide for thecarbonyl, by reversing the peptide bond, or by substituting a methylenefor the carbonyl group. Such modifications can be made by standardprocedures known to those skilled in the art. See, e.g., Spatola, A. F.,“Peptide Backbone Modifications: A Structure-Activity Analysis ofPeptides Containing Amide Bond Surrogates, Conformational Constraints,and Related Backbone Replacements,” in Chemistry and Biochemistry ofAmino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein(ed.), Marcel Dekker, Inc., New York (1983).

An analog is also meant to include polypeptides in which one or more ofthe amino acid residues include a substituent group, or polypeptideswhich are fused with another compound, e.g., a compound to increase thehalf-life of the polypeptide, e.g., polyethylene glycol.

By fragment is meant some portion of the naturally occurring LBPpolypeptide. Preferably, the fragment is at least about 100 amino acidresidues, more preferably at least about 50 amino acid residues, morepreferably yet at least about 30 amino acid residues, more preferablyyet at least about 20 amino acid residues, more preferably yet at leastabout 5 amino acid residues, more preferably yet at least about 4 aminoacid residues, more preferably yet at least about 3 amino acid residues,and most preferably at least about 2 amino acid residues in length.Fragments include, e.g., truncated secreted forms, proteolyticfragments, splicing fragments, other fragments, and chimeric constructsbetween at least a portion of the relevant gene, e.g., LBP-1, LBP-2 orLBP-3, and another molecule. Fragments of LBP can be generated bymethods known to those skilled in the art. In certain embodiments, thefragment is biologically active. The ability of a candidate fragment toexhibit a biological activity of LBP can be assessed by methods known tothose skilled in the art. For example, LBP fragments can be tested fortheir ability to bind to LDL and/or to an arterial extracellular matrixstructural component, as described herein. Also included are LBPfragments containing residues that are not required for biologicalactivity of the fragment or that result from alternative mRNA splicingor alternative protein processing events.

Fragments of a protein can be produced by any of a variety of methodsknown to those skilled in the art, e.g., recombinantly, by proteolyticdigestion, or by chemical synthesis. Internal or terminal fragments of apolypeptide can be generated by removing one or more nucleotides fromone end (for a terminal fragment) or both ends (for an internalfragment) of a nucleic acid which encodes the polypeptide. Expression ofthe mutagenized DNA produces polypeptide fragments. Digestion with“end-nibbling” endonucleases can thus generate DNAs which encode anarray of fragments. DNAs which encode fragments of a protein can also begenerated; e.g., by random shearing, restriction digestion or acombination of the above-discussed methods. For example, fragments ofLBP can be made by expressing LBP DNA which has been manipulated invitro to encode the desired fragment, e.g., by restriction digestion ofany of the DNA sequences of FIGS. 2A, 7A, 8A, 10-8 (SEQ ID NOS: 10-18,45, 46, and 48).

Fragments can also be chemically synthesized using techniques known inthe art, e.g., conventional Merrifield solid phase f-Moc or t-Bocchemistry for example, peptides of the present invention can bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

An LBP or a biologically active fragment or analog thereof, or a bindingmolecule or a biologically active fragment or analog thereof, can, e.g.,compete with its cognate molecule for the binding site on thecomplementary molecule, and thereby reduce or eliminate binding betweenLBP and the cellular binding molecule. LBP or a binding molecule can beobtained, e.g., from purification or secretion of naturally occurringLBP or binding molecule, from recombinant LBP or binding molecule, orfrom synthesized LBP or binding molecule.

Therefore, methods for generating analogs and fragments and testing themfor activity are known to those skilled in the art.

An agent can also be a nucleic acid used as an antisense molecule.Antisense therapy is meant to include, e.g., administration or in situgeneration of oligonucleotides or their derivatives which specificallyhybridize, e.g., bind, under cellular conditions, with the cellular mRNAand/or genomic DNA encoding an LBP polypeptide, or mutant thereof, so asto inhibit expression of the encoded protein, e.g., by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix.

In certain embodiments, the antisense construct binds to anaturally-occurring sequence of an LBP gene which, e.g., is involved inexpression of the gene. These sequences include, e.g., promoter, startcodons, stop codons, and RNA polymerase binding sites. In otherembodiments, the antisense construct binds to a nucleotide sequencewhich is not present in the wild type gene. For example, the antisenseconstruct can bind to a region of an LBP gene which contains aninsertion of an exogenous, non-wild type sequence. Alternatively, theantisense construct can bind to a region of an LBP gene which hasundergone a deletion, thereby bringing two regions of the gene togetherwhich are not normally positioned together and which, together, create anon-wild type sequence. When administered in vivo to a subject,antisense constructs which bind to non-wild type sequences provide theadvantage of inhibiting the expression of a mutant LBP gene, withoutinhibiting expression of any wild type LBP gene.

An antisense construct of the present invention can be delivered, e.g.,as an expression plasmid which, when transcribed in the cell, producesRNA which is complementary to at least a unique portion of the cellularmRNA which encodes an LBP polypeptide. An alternative is that theantisense construct is an oligonucleotide which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA (duplexing) and/or genomic sequences(triplexing) of an LBP gene. Such oligonucleotides are preferablymodified oligonucleotides which are resistant to endogenous nucleases,e.g. exonucleases and/or endonucleases, and are therefore stable invivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate,phosphorodithioates and methylphosphonate analogs of DNA and peptidenucleic acids (PNA). (See also U.S. Pat. Nos. 5,176,996; 5,264,564; and5,256,775). Additionally, general approaches to constructing oligomersuseful in antisense therapy have been reviewed. (See, e.g., Van der Krolet al., Biotechniques 6: 958-976, (1988); Stein et al., Cancer Res. 48:2659-2668 (1988)).

By mimetic is meant a molecule which resembles in shape and/or chargedistribution LBP or a binding molecule. The mimetic can be a peptide ora non-peptide. Mimetics can act as therapeutic agents because they can,e.g., competitively inhibit binding of LBP to a binding molecule. Byemploying, e.g., scanning mutagenesis, e.g., alanine scanningmutagenesis, linker scanning mutagenesis or saturation mutagenesis, tomap the amino acid residues of a particular LBP polypeptide involved inbinding a binding molecule, peptide mimetics, e.g., diazepine orisoquinoline derivatives, can be generated which mimic those residues inbinding to a binding molecule, and which therefore can inhibit bindingof the LBP to a binding molecule and thereby interfere with the functionof LBP. Non-hydrolyzable peptide analogs of such residues can begenerated using, e.g., benzodiazepine (see, e.g., Freidinger et al., inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands (1988)); azepine (see, e.g., Huffman et al., inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands (1988)); substituted gamma lactam rings (see, e.g.,Garvey et al., in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands (1988)); keto-methylenepseudopeptides (see, e.g., Ewenson et al., J. Med. Chem. 29: 295 (1986);Ewenson et al., in Peptides: Structure and Function (Proceedings of the9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.(1985)); β-turn dipeptide cores (see, e.g., Nagai et al., TetrahedronLett. 26: 647 (1985); Sato et al., J. Chem. Soc. Perkin Trans. 1: 1231(1986)); or β-aminoalcohols (see, e.g., Gordon et al., Biochem. Biophys.Res. Commun. 126:419 (1985); Dann et al., Biochem. Biophys. Res. Commun.134:71 (1986)).

Antibodies are meant to include antibodies against any moiety thatdirectly or indirectly affects LBP metabolism. The antibodies can bedirected against, e.g., LBP or a binding molecule, or a subunit orfragment thereof. For example, antibodies include anti-LBP-1, LBP-2 orLBP-3 antibodies; and anti-binding molecule antibodies. Antibodyfragments are meant to include, e.g., Fab fragments, Fab′ fragments,F(ab′)₂ fragments, F(v) fragments, heavy chain monomers, heavy chaindimers, heavy chain trimers, light chain monomers, light chain dimers,light chain trimers, dimers consisting of one heavy and one light chain,and peptides that mimic the activity of the anti-LBP or anti-bindingmolecule antibodies. For example, Fab₂′ fragments of the inhibitoryantibody can be generated through, e.g., enzymatic cleavage. Bothpolyclonal and monoclonal antibodies can be used in this invention.Preferably, monoclonal antibodies are used. Natural antibodies,recombinant antibodies or chimeric-antibodies, e.g., humanizedantibodies, are included in this invention. Preferably, humanizedantibodies are used when the subject is a human. Most preferably, theantibodies have a constant region derived from a human antibody and avariable region derived from an inhibitory mouse monoclonal antibody.Production of polyclonal antibodies to LBP is described in Example 6.Monoclonal and humanized antibodies are generated by standard methodsknown to those skilled in the art. Monoclonal antibodies can beproduced, e.g., by any technique which provides antibodies produced bycontinuous cell lines cultures. Examples include the hybridoma technique(Kohler and Milstein, Nature 256: 495-497 (1975), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today4:72 (1983)), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, A. R. Lisa, Inc., pp. 77-96 (1985)). Preferably, humanizedantibodies are raised through conventional production and harvestingtechniques (Berkower, I., Curr. Opin. Biotechnol. 7:622-628 (1996);Ramharayan and Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)). Incertain preferred embodiments, the antibodies are raised against theLBP, preferably the LDL-binding site, and the Fab fragments produced.These antibodies, or fragments derived therefrom, can be used, e.g., toblock the LDL-binding sites on the LBP molecules.

Agents also include inhibitors of a molecule that are required forsynthesis, post-translational modification, or functioning of LBP.and/or a binding molecule, or activators of a molecule that inhibits thesynthesis or functioning of LBP and/or the binding molecule. Agentsinclude, e.g., cytokines, chemokines, growth factors, hormones,signaling components, kinases, phosphatases, homeobox proteins,transcription factors, editing factors, translation factors andpost-translation factors or enzymes. Agents are also meant to includeionizing radiation, non-ionizing radiation, ultrasound and toxic agentswhich can, e.g., at least partially inactivate or destroy LBP and/or thebinding molecule.

An agent is also meant to include an agent which is not entirely LBPspecific. For example, an agent may alter other genes or proteinsrelated to arterial plaque formation. Such overlapping specificity mayprovide additional therapeutic advantage.

The invention also includes the agent so identified as being useful intreating atherosclerosis.

The invention also includes a method for evaluating an agent for theability to alter the binding of LBP polypeptide to a binding molecule.An agent is provided. An LBP polypeptide is provided. A binding moleculeis provided. The agent, LBP polypeptide and binding molecule arecombined. The formation of a complex comprising the LBP polypeptide andbinding molecule is detected. An alteration in the formation of thecomplex in the presence of the agent as compared to in the absence ofthe agent is indicative of the agent altering the binding of the LBPpolypeptide to the binding molecule.

In preferred embodiments, the LBP polypeptide is LBP-1, LBP-2 or LBP-3.Examples of a binding molecule include native LDL, modified LDL, e.g.,methylated LDL or oxidized LDL, and arterial extracellular matrixstructural components.

Altering the binding includes, e.g., inhibiting or promoting thebinding. The efficacy of the agent can be assessed, e.g., by generatingdose response curves from data obtained using various concentrations ofthe agent. Methods for determining formation of a complex are standardand are known to those skilled in the art, e.g., affinitycoelectrophoresis (ACE) assays or ELISA assays as described herein.

The invention also includes the agent so identified as being able toalter the binding of an LBP polypeptide to a binding molecule.

The invention also includes a method for evaluating an agent for theability to bind to an LBP polypeptide. An agent is provided. An LBPpolypeptide is provided. The agent is contacted with the LBPpolypeptide. The ability of the agent to bind to the LBP polypeptide isevaluated. Preferably, the LBP polypeptide is LBP-1, LBP-2 or LBP-3.Binding can be determined, e.g., by measuring formation of a complex bystandard methods known to those skilled in the art, e.g., affinitycoelectrophoresis (ACE) assays or ELISA assays as described herein.

The invention also includes the agent so identified as being able tobind to LBP polypeptide.

The invention also includes a method for evaluating an agent for theability to bind to a nucleic acid encoding an LBP regulatory sequence.An agent is provided. A nucleic acid encoding an LBP regulatory sequenceis provided. The agent is contacted with the nucleic acid. The abilityof the agent to bind to the nucleic acid is evaluated. Preferably, theLBP regulatory sequence is an LBP-1, LBP-2 or LBP-3 regulatory sequence.Binding can be determined, e.g., by measuring formation of a complex bystandard methods known to those skilled in the art, e.g., DNA mobilityshift assays, DNase I footprint analysis Molecular Biology, Theinvention being able to bind sequence. (Ausubel et al., ed., CurrentProtocols in John Wiley & Sons, New York, N.Y., (1989)).

The invention also includes the agent so identified as to a nucleic acidencoding an LBP regulatory sequence.

The invention also includes a method for treating atherosclerosis in ananimal. An animal in need of treatment for atherosclerosis is provided.An agent capable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the animal in a therapeuticallyeffective amount such that treatment of the atherosclerosis occurs.

In certain preferred embodiments, the agent is an LBP polypeptide, e.g.,LBP-1, LBP-2 or LBP-3, or a biologically active fragment or analogthereof. The agent can be, e.g., the polypeptide as set forth in SEQ IDNOS: 1-9, 43, 44, and 47. Preferably, the agent is a polypeptide of nomore than about 100 amino acid residues in length, more preferably of nomore than about 50 amino acid residues, more preferably yet of no morethan about 30 amino acid residues, more preferably yet of no more thanabout 20 amino acid residues, more preferably yet of no more than about10 amino acid residues, more preferably yet of no more than about 5amino acid residues, more preferably yet of no more than about 4 aminoacid residues, more preferably yet of no more than about 3 amino acidresidues, and most preferably of no more than about 2 amino acidresidues. Preferably, the polypeptide includes at least about 20% acidicamino acid residues, more preferably yet at least about 40% acidic aminoacid residues, more preferably yet at least about 60% acidic amino acidresidues, more preferably yet at least about 80% acidic amino acidresidues, more preferably yet at least about 90% acidic amino acidresidues, more preferably yet at least about 95% acidic amino acidresidues, and most preferably at least about 98% acidic amino acidresidues. Acidic amino acid residues include aspartic acid and glutamicacid. An example of such an LBP poly-peptide is BHF-1, which is a 20amino acid length fragment of human or rabbit LBP-1 which contains aminoacid residues 14 through 33. See FIG. 9 (SEQ ID NO: 9). 45% of the aminoacid residues of BHF-1 are acidic. The invention also includesbiologically active fragments and analogs of BHF-1.

Other preferred acidic regions from the LBPs are amino acid residues 329through 343 (SEQ ID NO: 19), 329 through 354 (SEQ ID NO: 20), 344through 354 (SEQ ID NO: 21), and 529 through 538 (SEQ ID NO: 22) ofhuman LBP-2 as depicted in FIG. 7A (SEQ ID NO: 43); amino acid residues14 through 43 (SEQ ID NO: 23)and 38 through 43 (SEQ ID NO: 24) of rabbitor human LBP-1 as depicted in FIG. 1 (SEQ ID NO: 1) and FIG. 6 (SEQ IDNO: 6); amino acid residues 338 through 353 (SEQ ID NO: 25), 338 through365 (SEQ ID NO: 26), 354 through 365 (SEQ ID NO: 27), and 444 through453 (SEQ ID NO: 28) of rabbit LBP-2 as depicted in FIG. 2A (SEQ ID NO:47); amino acid residues 96 through 110 (SEQ ID NO: 29) of rabbit LBP-3as depicted in FIG. 5 (SEQ ID NO: 5); and amino acid residues 69-75 (SEQID NO: 41) of human LBP-3 as depicted in FIG. 8A (SEQ ID NO: 44). Theinvention is also meant to include biologically active fragments andanalogs of any of these polypeptides.

Other examples of agents include homopolymers and heteropolymers of anyamino acid or amino acid analog. In certain preferred embodiments, theagent is a homopolymer of an acidic amino acid or analog thereof. Incertain embodiments, the agent is a heteropolymer of one or more acidicamino acids and one or more other amino acids, or analogs thereof. Forexample, agents include poly(glu), poly(asp), poly(glu asp), poly(gluN), poly (asp N) and poly(glu asp N). By N is meant any amino acid, oranalog thereof, other than glu or asp. By poly(glu asp) is meant allpermutations of glu and asp for a given length peptide. A preferredpeptide is poly(glu) of no more than about 10 amino acids in length,preferably about 7 amino acids in length.

In certain preferred embodiments, the agent is an LBP nucleic acid or abiologically active fragment or analog thereof, e.g., a nucleic acidencoding LBP-1, LBP-2 or LBP-3 polypeptide, or a biologically activefragment or analog thereof. The agent can be, e.g., a nucleic acidcomprising a nucleotide sequence as set forth in SEQ ID NOS: 10-18, 45,46, and 48. In other embodiments, the agent is an antisense molecule,e.g., one which can bind to an LBP gene sequence.

Treating is meant to include, e.g., preventing, treating, reducing thesymptoms of, or curing the atherosclerosis. Administration of the agentcan be accomplished by any method which allows the agent to reach thetarget area, e.g., a target cell or the extracellular matrix. Thesemethods include, e.g., injection, deposition, implantation,suppositories, oral ingestion, inhalation, topical administration, orany other method of administration where access to the target area bythe agent is obtained. Injections can be, e.g., intravenous,intradermal, subcutaneous, intramuscular or intraperitoneal.Implantation includes inserting implantable drug delivery systems, e.g.,microspheres, hydrogels, polymeric reservoirs, cholesterol matrices,polymeric systems, e.g., matrix erosion and/or diffusion systems andnon-polymeric systems, e.g., compressed, fused or partially fusedpellets. Suppositories include glycerin suppositories. Oral ingestiondoses can be enterically coated. Inhalation includes administering theagent with an aerosol in an inhalator, either alone or attached to acarrier that can be absorbed.

Administration of the agent can be alone or in combination with othertherapeutic agents. In certain embodiments, the agent can be combinedwith a suitable carrier, incorporated into a liposome, or incorporatedinto a polymer release system.

In certain embodiments of the invention, the administration can bedesigned so as to result in sequential exposures to the agent over sometime period, e.g., hours, days, weeks, months or years. This can beaccomplished by repeated administrations of the agent by one of themethods described above, or alternatively, by a controlled releasedelivery system in which the agent is delivered to the animal over aprolonged period without repeated administrations. By a controlledrelease delivery system is meant that total release of the agent doesnot occur immediately upon administration, but rather is delayed forsome time. Release can occur in bursts or it can occur gradually andcontinuously. Administration of such a system can be, e.g., by longacting oral dosage forms, bolus injections, transdermal patches orsubcutaneous implants.

Examples of systems in which release occurs in bursts include, e.g.,systems in which the agent is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive to aspecific stimulus, e.g., temperature, pH, light, magnetic field, or adegrading enzyme, and systems in which the agent is encapsulated by anionically-coated microcapsule with a microcapsule core-degrading enzyme.Examples of systems in which release of the agent is gradual andcontinuous include, e.g., erosional systems in which the agent iscontained in a form within a matrix, and diffusional systems in whichthe agent permeates at a controlled rate, e.g., through a polymer. Suchsustained release systems can be, e.g., in the form of pellets orcapsules.

The agent can be suspended in a liquid, e.g., in dissolved form orcolloidal form. The liquid can be a solvent, partial solvent ornon-solvent. In many cases water or an organic liquid can be used.

The agent can be administered prior to or subsequent to the appearanceof atherosclerosis symptoms. In certain embodiments, the agent isadministered to patients with familial histories of atherosclerosis, orwho have phenotypes that may indicate a predisposition toatherosclerosis, or who have been diagnosed as having a genotype whichpredisposes the patient to atherosclerosis, or who have other riskfactors, e.g., hypercholesterolemia, hypertension or smoking.

The agent is administered to the animal in a therapeutically effectiveamount. By therapeutically effective amount is meant that amount whichis capable of at least partially preventing or reversingatherosclerosis. A therapeutically effective amount can be determined onan individual basis and will be based, at least in part, onconsideration of the species of animal, the animal's size, the animal'sage, the agent used, the type of delivery system used, the time ofadministration relative to the onset of atherosclerosis symptoms, andwhether a single, multiple, or controlled release dose regimen isemployed. A therapeutically effective amount can be determined by one ofordinary skill in the art employing such factors and using no more thanroutine experimentation.

Preferably, the concentration of the agent is at a dose of about 0.1 toabout 1000 mg/kg body weight/day, more preferably at about 0.1 to about500 mg/kg/day, more preferably yet at about 0.1 to about 100 mg/kg/day,and most preferably at about 0.1 to about 5 mg/kg/day. The specificconcentration partially depends upon the particular agent used, as someare more effective than others. The dosage concentration of the agentthat is actually administered is dependent at least in part upon thefinal concentration that is desired at the site of action, the method ofadministration, the efficacy of the particular agent, the longevity ofthe particular agent, and the timing of administration relative to theonset of the atherosclerosis symptoms. Preferably, the dosage form issuch that it does not substantially deleteriously affect the animal. Thedosage can be determined by one of ordinary skill in the art employingsuch factors and using no more than routine experimentation.

In certain embodiments, various gene constructs can be used as part of agene therapy protocol to deliver nucleic acids encoding an agent, e.g.,either an agonistic or antagonistic form of an LBP polypeptide. Forexample, expression vectors can be used for in vivo transfection andexpression of an LBP polypeptide in particular cell types so as toreconstitute the function of, or alternatively, abrogate the functionof, LBP polypeptide in a cell in which non-wild type LBP is expressed.Expression constructs of the LBP polypeptide, and mutants thereof, maybe administered in any biologically effective carrier, e.g., anyformulation or composition capable of effectively delivering the LBPgene to cells in vivo. Approaches include, e.g., insertion of thesubject gene in viral vectors including, e.g., recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors infect ortransduce cells directly; plasmid DNA can be delivered with the help of,for example, cationic liposomes (lipofectin™ (Life Technologies, Inc.,Gaithersburg, Md.) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or Ca₃,(P0₄)₂ precipitation carried out in vivo. Theabove-described methods are known to those skilled in the art and can beperformed without undue experimentation. Since transduction ofappropriate target cells represents the critical first step in genetherapy, choice of the particular gene delivery system will depend onsuch factors as the phenotype of the intended target and the route ofadministration, e.g., locally or systemically. Administration can bedirected to one or more cell types, and to one or more cells within acell type, so as to be therapeutically effective, by methods that areknown to those skilled in the art. In a preferred embodiment, the agentis administered to arterial wall cells of the animal. For example, agenetically engineered LBP gene is administered to arterial wall cells.In certain embodiments, administration is done in a prenatal animal orembryonic cell. It will be recognized that the particular gene constructprovided for in vivo transduction of LBP expression is also useful forin vitro transduction of cells, such as for use in the diagnostic assaysdescribed herein.

In certain embodiments, therapy of atherosclerosis is performed withantisense nucleotide analogs of the genes which code for the LBPs.Preferably, the antisense nucleotides have non-hydrolyzable “backbones,”e.g., phosphorothioates, phosphorodithioates or methylphosphonates. Thenucleoside base sequence is complementary to the sequence of a portionof the gene coding for, e.g., LBP-1, 2 or 3. Such a sequence might be,e.g., ATTGGC if the gene sequence for the LBP is TAACCG. One embodimentof such therapy would be incorporation of an antisense analog of aportion of one of the LBP genes in a rslowrelease medium, e.g.,polyvinyl alcohol, which is administered, e.g., by subcutaneousinjection, so as to release the antisense nucleotide analog over aperiod of weeks or months. In another embodiment, the antisense analogis incorporated into a polymeric matrix, e.g., polyvinyl alcohol, suchthat the gel can be applied locally to an injured arterial wall toinhibit LBP synthesis and prevent LDL accumulation, e.g., afterangioplasty or atherectomy.

The invention also includes a method for treating an animal at risk foratherosclerosis. An animal at risk for atherosclerosis is provided. Anagent capable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the animal in a therapeuticallyeffective amount such that treatment of the animal occurs. Being at riskfor atherosclerosis can result from, e.g., a family history ofatherosclerosis, or phenotypic symptoms which predispose toatherosclerosis, e.g., having hypercholesterolemia, hypertension orsmoking.

The invention also includes a method for treating a cell having anabnormality in structure or metabolism of LBP. A cell having anabnormality in structure or metabolism of LBP is provided. An agentcapable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the cell in a therapeuticallyeffective amount such that treatment of the cell occurs.

In certain embodiments, the cell is obtained from a cell culture ortissue culture or an embryo fibroblast. The cell can be, e.g., part ofan animal, e.g., a natural animal or a nonhuman transgenic animal.Preferably, the LBP is LBP-1, LBP-2 or LBP-3.

The invention also includes a pharmaceutical composition for treatingatherosclerosis in an animal comprising a therapeutically effectiveamount of an agent, the agent being capable of altering an aspect of LBPmetabolism or structure in the animal so as to result in treatment ofthe atherosclerosis, and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include, e.g., saline, liposomesand lipid emulsions.

In certain preferred embodiments, the agent of the pharmaceuticalcomposition is an LBP polypeptide, e.g., LBP-1, LBP-2 or LBP-3, or abiologically active fragment or analog thereof. The agent can be, e.g.,the polypeptide as set forth in SEQ ID NOS: 1-9, 43, 44, and 47.Preferably, the agent is a polypeptide of no more than about 100 aminoacid residues in length, more preferably of no more than about 50 aminoacid residues, more preferably yet of no more than about 30 amino acidresidues, more preferably yet of no more than about 20 amino acidresidues, more preferably yet of no more than about 10 amino acidresidues, more preferably yet of no more than about 5 amino acidresidues, more preferably yet of no more than about 4 amino acidresidues, more preferably yet of no more than about 3 amino acidresidues, and most preferably of no more than about 2 amino acidresidues. Preferably, the polypeptide includes at least about 20% acidicamino acid residues, more preferably yet at least about 40% acidic aminoacid residues, more preferably yet at least about 60% acidic amino acidresidues, more preferably yet at least about 80% acidic amino acidresidues, more preferably yet at least about 90% acidic amino acidresidues, more preferably yet at least about 95% acidic amino acidresidues, and most preferably at least about 98%acidic amino acidresidues.

In certain preferred embodiments, the agent is an LBP nucleic acid,e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or abiologically active fragment or analog thereof. The agent can be, e.g.,a nucleic acid comprising a nucleotide sequence as set forth in SEQ IDNOS: 10-18, 45, 46, and 48.

The invention also includes a vaccine composition for treatingatherosclerosis in an animal comprising a therapeutically effectiveamount of an agent, the agent being capable of altering an aspect of LBPmetabolism or structure in the animal so as to result in treatment ofthe atherosclerosis, and a pharmaceutically acceptable carrier.

The invention also includes a method for diagnosing atheroscleroticlesions in an animal. An animal is provided. A labeled agent capable ofbinding to LBP present in atherosclerotic lesions is provided. Thelabeled agent is administered to the animal under conditions which allowthe labeled agent to interact with the LBP so as to result in labeledLBP. The localization or quantification of the labeled LBP is determinedby imaging so as to diagnose the presence of atherosclerotic lesions inthe animal.

Preferably, the LBP is LBP-1, LBP-2 or LBP-3. The imaging can beperformed by standard methods known to those skilled in the art,including, e.g., magnetic resonance imaging, gamma camera imaging,single photon emission computed tomographic (SPECT) imaging, or positronemission tomography (PET).

Preferably, agents that bind tightly to LBPs in atherosclerotic lesionsare used for atherosclerotic imaging and diagnosis. The agent isradiolabeled with, e.g., ^(99m)Tc or another isotope suitable forclinical imaging by gamma camera, SPECT, PET scanning or other similartechnology. Since LBPs occur in very early lesions, such imaging is moresensitive than angiography or ultrasound for locating very early lesionswhich do not yet impinge on the arterial lumen to cause a visible bulgeor disturbed flow. In addition to locating both early and more developedlesions, the imaging agents which bind to LBPs can also be used tofollow the progress of atherosclerosis, as a means of evaluating theeffectiveness of both dietary and pharmacological treatments.

Thus, a diagnostic embodiment of the invention is the adaptation of,e.g., a peptide complementary to one of the LBPs, by radiolabeling itand using it as an injectable imaging agent for detection of occultatherosclerosis. The peptide is selected from those known to bind toLBPs, e.g., RRRRRRR (SEQ ID NO:52) or KKLKLXX (SEQ ID NO:53), or anyother polycationic peptide which binds to the highly electronegativedomains of the LBPs. For extracorporeal detection with a gammascintillation (Anger) camera, technetium-binding ligands, e.g., CGC,GGCGC, or GGCGCF, can be incorporated into the peptides at theN-terminus or C-terminus for ^(99m)Tc labeling. For external imaging bymagnetic resonance imaging (MRI), e.g., the gadolinium-binding chelator,diethylene triamine penta-acetic acid (DTPA), is covalently bound to theN- or C-terminus of the peptides. In yet other embodiments, theLBP-binding peptides are covalently bound, e.g., to magnetic ion oxideparticles by standard methods known to those skilled in the art, e.g.,conjugating the peptides with activated polystyrene resin beadscontaining magnetic ion oxide.

The invention also includes a method for immunizing an animal against anLBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or analog thereof. Ananimal having LDL is provided. An LBP or fragment or analog thereof isprovided. The LBP or fragment or analog thereof is administered to theanimal so as to stimulate antibody production by the animal to the LBPor fragment or analog thereof such that binding of the LBP to the LDL isaltered, e.g., decreased or increased.

The invention also includes a method of making a fragment or analog ofLBP polypeptide, the fragment or analog having the ability to bind tomodified LDL and native LDL. An LBP polypeptide is provided. Thesequence of the LBP polypeptide is altered. The altered LBP polypeptideis tested for the ability to bind to modified LDL, e.g., methylated LDL,oxidized LDL, acetylated LDL, cyclohexanedione-treated LDL (CHD-LDL),and to native LDL.

The fragments or analogs can be generated and tested for their abilityto bind to these modified LDLs and to native LDL, by methods known tothose skilled in the art, e.g., as described herein. Preferably, theyare tested for their ability to bind to methylated LDL and native LDL.The binding activity of the fragment or analog can be greater or lessthan the binding activity of the native LBP. Preferably, it is greater.In preferred embodiments, the LBP is LBP-1, LBP-2 or LBP-3.

The invention also includes a method for isolating a cDNA encoding anLBP. A cDNA library is provided. The cDNA library is screened for a cDNAencoding a polypeptide which binds to native LDL and modified LDL, e.g.,methylated LDL or oxidized LDL. The cDNA which encodes this polypeptideis isolated, the cDNA encoding an LBP.

Atherosclerosis in a hyperlipidemic subject can be reduced following thegeneration of an immune response in the subject by immunization withLBPs. Numerous immunotherapeutic products can be used to generateantibodies that will block the binding between LDL and LBPs.

The injection of one or more LBPs can result in the production ofanti-LBP antibodies, resulting in a reduction in ,e.g., aorticatherosclerosis. This effect is thought to be mediated by an inhibitionof LBP binding to LDL. LBP immunogens that can be used in the inventioninclude human LBPs, non-human LBPs, recombinant LBPs, and proteinsstructurally related to the LBPs described herein, e.g. non-naturallyoccurring proteins that differ from a naturally occurring LBP at one ormore amino acid residues. In addition to full length proteins, injectingone or more peptides that include an LBP domain can generate aneffective immune response. For example, the injection of a peptidecomprising an LBP domain having LDL-binding activity can cause anorganism to make antibodies to the LBP binding sites for LDL. Thesepeptide immunogens can include sequences derived from human LBPs,non-human LBPs, recombinant LBPs, and proteins structurally related tothe LBPs described herein.

Modifications can be made to a protein or peptide immunogen of theinvention to increase its immunogenicity. The immunogen can beconjugated or coupled with a carrier, e.g. a Cholera toxin B chain ormonoclonal antibodies. The immunogen can be precipitated with aluminumsalts or cross-linked with formaldehyde or other aldehydes. The proteinmay be mixed with a physiologically acceptable diluent such as water,phosphate buffered saline, or saline. The composition may furtherinclude an adjuvant. In addition to RIBI adjuvant, adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide areall well known in the art. Adjustments in the adjuvant of the inventioncan be made to affect the immunogenicity of the peptide or protein.Examples of such modifications include using: aluminum salts; cytokines;MF59 (microfluidized emulsion of oil and surfactants); SAF-1 (oil-basedemulsion); saponin derivatives; polymers (such as polyphosphazene); andbacterial toxins. Additional descriptions of antigenic protein-adjuvantcombinations are described in WO 99/54452 (herein incorporated byreference) and WO 99/49890 (herein incorporated by reference).

In addition to delivery of the proteins and peptides described above,numerous other delivery systems can be used to generate theanti-atherosclerotic immunity of the invention. The LBP immunogen can bedelivered either directly as a protein antigen or alternatively as anucleic acid that encodes the protein antigen. The immunotherapeuticproducts of the invention, either protein or nucleic acid, can bedelivered by numerous delivery routes. These include injection,deposition, implantation, suppositories, oral ingestion, inhalation(e.g., delivery via a nasal spray), and topical administration (e.g.,delivery via a skin patch).

A nucleic acid encoding an immunogen of the invention can be directlyadministered, for example by injection, to tissues and expressed as aprotein. The DNA or RNA can be either associated with a delivery vehicle(e.g., viruses, bacteria, liposomes, and gold beads) or naked (free fromassociation with transfection-facilitating proteins, viral particles,liposomal formulations, charged lipids and calcium phosphateprecipitating). The nucleic acid can optionally include a promoter, e.g.a viral promoter. The immunogen encoded by the nucleic acid is producedin the host, resulting in the generation of an immune response. Methodsfor the delivery of nucleic acid sequences encoding therapeutic proteinsand peptides are described in detail by Felgner et al. (U.S. Pat. No.5,580,859; herein incorporated by reference) and Barbet et al. (U.S.Pat. No. 6,025,338; herein incorporated by reference). Vaccinecompositions of viral liposomes comprising a nucleic acid, e.g. an RNA,encoding a protein antigen are described in WO 99/52503 (hereinincorporated by reference). Proteins and nucleic acids encoding peptidescan also be delivered to an individual by their encapsulation inliposomes, microparticles, and ISCOMS, all of which are well known inthe art (see, e.g., U.S. Pat. No. 6,013,258, herein incorporated byreference).

A nucleic acid encoding an immunogen of the invention can also beincluded in the genome of a plant, so as to result in the production ofthe immunogen by plant tissues. The genetically modified plant may thenconsumed by an individual, resulting in the ingestion of the immunogenand the generation of an anti-LBP immune response. Methodology for thegeneration and usage of edible plant vaccines is described in WO99/54452 (herein incorporated by reference).

Numerous plants may be useful for the production of an edible vaccine,including: tobacco, tomato, potato, eggplant, pepino, yam, soybean, pea,sugar beet, lettuce, bell pepper, celery, carrot, asparagus, onion,grapevine, muskmelon, strawberry, rice, sunflower, rapeseed/canola,wheat, oats, maize, cotton, walnut, spruce/conifer, poplar and apple.The edible vaccine can include a plant cell transformed with a nucleicacid construct comprising a promoter and a sequence encoding an LBP. Thesequence may optionally encode a chimeric protein, comprising a choleratoxin subunit B peptide fused to the LBP peptide. Preferred plantpromoters of the invention include CaMV 35S, patatin, mas, andgranule-bound starch synthase promoters. Additional useful promoters andenhancers are described in WO 99/54452.

The edible vaccine of the invention can be administered to a mammalsuffering from or at risk of atherosclerosis. Preferably, an ediblevaccine is administered orally, e.g. consuming a transgenic plant of theinvention. The transgenic plant can be in the form of a plant part,extract, juice, liquid, powder, or tablet. The edible vaccine can alsobe administered via an intranasal route.

Microorganisms, e.g., attenuated viruses or bacteria, can be used in theinvention by including a nucleic acid encoding an LBP immunogen in thegenome of the microorganism. This modified vector can then be deliveredto a host, resulting in the in vivo production of the immunogen. Theimmune response generated by these vectors is expected to result inanti-atherosclerotic immunity. Nucleic acid molecules are inserted intomicroorganism genomes by standard methods known in the art (U.S. Pat.No. 5,866,136 and U.S. Pat. No. 6,025,164, both of which are hereinincorporated by reference) The anti-atherosclerotic methods of theinvention are directed to treating a subject, e.g., a human, primate,horse, dog, cat, or goat, at risk for atherosclerosis by stimulating ananti-LBP response in the subject by immunotherapy. The LBP proteins andpeptides of the invention may be delivered to the subject by thenumerous delivery systems described herein. The immunotherapy maycomprise an initial immunization followed by additional, e.g. one, two,or three, boosters.

The invention also includes a method of treating a subject at risk foratherosclerosis by (1) providing a subject at risk for atherosclerosisand (2) administering to the subject one or more of the following: (a)an LBP protein or fragment or analog thereof and an adjuvant; (b) anucleic acid encoding an LBP protein; (c) a virus or bacteria comprisinga nucleic acid encoding an LBP protein; and (d) an edible plantcomprising a nucleic acid encoding an LBP protein. The LBP protein usedin this method can be any LBP described herein, e.g., LBP-1, LBP-2, orLBP-3. A combination of more than one nucleic acid or LBP protein orfragment or analog thereof can be administered to the subject. Forexample, combinations of LBP proteins, or nucleic acids encoding LBPproteins, include: (1) LBP-1 and LBP-2; (2) LBP-1 and LBP-3; (3) LBP-2and LBP-3; and (4) LBP-1, LBP-2, and LBP-3. This method optionallyincludes a step of diagnosing the subject as being at risk foratherosclerosis.

Also provided by the invention is a method of treating a subject at riskfor atherosclerosis whereby a non-autologous LBP protein or a nucleicacid encoding a non-autologous LBP protein is delivered to the subjectto generate an immune response to an autologous LBP. Specifically, thismethod entails identifying one or more autologous LBP proteins, e.g.,LBP-1, LBP-2, or LBP-3, produced by the subject. The identification canby, e.g., DNA sequence analysis, protein sequence analysis, antibodyreactivity, hybridization analysis, or nucleic acid amplification. Next,a non-autologous LBP protein, e.g., allogeneic, xenogeneic, or agenetically modified, non-naturally occurring protein that differs atone or more amino acid residues from the one or more LBP proteins, isadministered to the subject. Alternatively, a nucleic acid encoding anon-autologous LBP protein is administered to the subject. Theanti-atherosclerotic effectiveness of this immunotherapeutic product isdetermined by its ability to induce an immune response against one ormore autologous LBP proteins when administered to the subject. It istherefore expected that extensive differences between a non-autologousand autologous LBP protein will not result in cross immunoreactivity.This method optionally includes a step of diagnosing the subject asbeing at risk for atherosclerosis.

Another method of the invention is a method of treating a subject atrisk for atherosclerosis by increasing the levels of one or more LBPproteins circulating in the plasma. According to this method, eitherautologous or non-autologous LBP levels may be increased. Non-autologousLBP proteins include, e.g., allogeneic LBP, xenogeneic LBP, andgenetically modified LBP. The plasma levels of one or more LBP proteinscan be increased by the delivery of a nucleic acid encoding an LBPprotein. Because LBP generally does not normally occur as a circulatingprotein, the endogenous molecule is expected to be susceptible to immunerecognition when delivered in a soluble form. This method optionallyincludes a step of diagnosing the subject as being at risk foratherosclerosis.

Also included in the invention is a pharmaceutical compositioncontaining one or more LBP proteins, e.g., LBP-1, LBP-2, or LBP-3, mixedwith an adjuvant, suitable for use in humans. The pharmaceuticalcomposition can contain a combination of more than one LBP protein. Forexample, compositions can include any of the following: (1) LBP-1 andLBP-2; (2) LBP-1 and LBP-3; (3) LBP-2 and LBP-3; and (4) LBP-1, LBP-2,and LBP-3.

Also included in the invention is a cell therapy system, whereby a cellexpressing an LBP is delivered to a subject at risk for atherosclerosis.This cell can be engineered to express either an autologous ornon-autologous LBP protein or peptide of the invention. Delivery of thisengineered cell to a subject results in the in vivo production of an LBPprotein and the associated immunotherapy produced when either theprotein or a nucleic acid encoding the protein is provided to anindividual. Cell therapy methods are described in U.S. Pat. No.5,955,095 (herein incorporated by reference).

The following non-limiting examples further illustrate the presentinvention.

EXAMPLES Example 1 Construction of a Rabbit cDNA Library

This example illustrates the construction of a rabbit cDNA library usingmRNA from balloon-deendothelialized healing rabbit abdominal aorta.Balloon-catheter deendothelialized rabbit aorta has been shown to be avalid model for atherosclerosis (Minick et al., Am. J. Pathol.95:131-158 (1979).

The mRNA was obtained four weeks after ballooning to maximize focal LDLbinding in the ballooned rabbit aorta. First strand cDNA synthesis wascarried out in a 50 μl reaction mixture containing 4 μg mRNA; 2 μg oligod(T)primer; methylation dNTP mix (10 mM each); 10 mM DTT; 800 unitssuperscript II RT (Life Technologies, Gaithersburg, Md.); 1× firststrand cDNA synthesis buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 5 mMMgCl₂), which was incubated for 1 hr at 37° C. The reaction mixture wasthen adjusted to 250 μl through the addition of 1× second strand buffer(30 mM Tris-HCl, pH 7.5; 105 mM _(KCl); 5.2 mM MgCl₂); 0.1 mM DTT;methylation dNTP mix (10 mM each); 50 units E. coli DNA polymerase 1, 3units RNase H; 15 units E. coli DNA ligase (all enzymes from LifeTechnologies), which was incubated for an additional 2.5 hr at 15° C.The resulting double-stranded cDNAs (dscDNA) were then treated with 1.5units T4 DNA polymerase (Novagen Inc., Madison, Wis.) for 20 min at 1 °°C. to make blunt-ended dscDNA. These were then concentrated by ethanolprecipitation and EcoRl/Hind III linkers were attached to the ends by T4DNA ligase (Novagen Inc.). The linker-ligated cDNAs were treated withEcoRl and Hind111 restriction enzymes to produce EcoRl and Hind IIIrecognition sequences at their 5′ and 3′ ends, respectively. After theremoval of linker DNA by gel exclusion chromatography, the dscDNAs wereinserted into λEXlox phage arms (Novagen Inc.) in a unidirectionalmanner by T4 DNA ligase and packaged into phage particles according tothe manufacturer's protocol (Novagen Inc.). A phage library of cDNAscontaining 2×10⁶ independent clones was established from 4 μg of mRNA.

Example 2 Identification of Rabbit cDNAs Encoding LDL Binding Proteins(LBPs)

This example illustrates a method of functionally screening a rabbitcDNA library so as to identify cDNAs encoding LBPs which bind to bothnative LDL and methyl LDL. Methyl LDL is not recognized by previouslyreported cell surface receptors. See, e.g., Weisgraber et al., J. Biol.Chem. 253:9053-9062 (1978).

A fresh overnight culture of E. coli ER1647 cells (Novagen Inc.) wasinfected with the cDNA phage obtained from Example 1, and plated at adensity of 2×10⁴ plaque-forming units (pfu) in 150 mm diameter platescontaining 2× YT agar. A total of 50 plates, equivalent to 1×10⁶ phage,were plated and incubated at 37° C. until the plaques reached 1 mm indiameter (5-6 hr). A dry nitrocellulose membrane, which had previouslybeen saturated with 10 mM IPTG solution, was layered on top of eachplate to induce the production of recombinant protein, as well as toimmobilize the proteins on the membranes. The plates were incubated at37° C. for an additional 3-4 hr, and then overnight at 4° C.

The next day, the membranes were lifted from each plate and processed asfollows. Several brief rinses in TBST solution (10 mM Tris-HCl, pH 8.0;150 mM NaCl, 0.05% Tween 20); two 10-min rinses with 6M guanidine-HCl inHBB (20 mM HEPES, pH 7.5; 5 mM MgCl₂, 1 mM DTT, and 5 mM KCl); two 5-minrinses in 3M guanidine-HCl in HBB; a final brief rinse in TBSEN (TBS, 1mM EDTA, 0.02% NaN₃).

The membranes were then blocked for 30 min at room temperature in asolution of TBSEN with 5% non-fat dry milk, followed by 10 min in TBSENwith 1% non-fat dry milk. Following blocking, the membranes wereincubated with native human LDL (obtained as described in Example 11 ormethylated human LDL (meLDL) (see Weisgraber et al., J. Biol. Chem.253:9053-9062 (1978)), at a concentration of 4 μg/ml, in a solutioncontaining 1×TBSEN, 1% non-fat dry milk, 1 mM PMSF, 0.5× proteaseinhibitor solution (1 mM ε-amino caproic acid/1 mM benzamidine).Incubation was for 4 hr at room temperature in a glass Petri dish withgentle stirring on a stirring table, followed by overnight at 4° C. withno stirring.

Specifically bound meLDL and native LDL were detected on thenitrocellulose membranes by antibodies against human LDL. Sheepanti-human LDL polyclonal antibodies (Boehringer Mannheim, Indianapolis,IN) were adsorbed with E. coli plys E cell extracts to abolishbackground. For adsorption, E. coli plys E cells were grown to logphase, spun down and resuspended in PBS containing 1 mM PMSF, 2 mMε-amino caproic acid, and 1 mM benzamidine. The cell suspension thenunderwent 8 freeze-thaw cycles via immersion in liquid nitrogen and coldrunning tap water, respectively. The anti LDL antibodies/cell extractsolution were incubated with gentle stirring for 1 hr at 4° C. (1 ml ofantibody solution/3 mg crude cell extract). Following incubation, themixture was presence of 0.02% NaN₃, until use. The membranes wereprocessed for immunoscreening as follows: (i) three 5-min washes at roomtemperature in TBSEN containing 1% gelatin; (ii) 30 min incubation inPBS, pH 7.4 with 1% gelatin; (iii) two-hr room temperature incubationwith gentle stirring in fresh PBS/gelatin solution containing adsorbedsheep anti-human LDL antibodies (Boehringer Manheim, Indianapolis, Ind.)(1: 1000 dilution); (iv) three brief washes in TBS, pH 7.4; (v) one-hrroom temperature incubation with gentle stirring in PBS/gelatin solutioncontaining donkey anti-sheep alkaline phosphatase-conjugated antibodies(Sigma, St. Louis, Mo.) (1: 10,000 dilution); (vi) three brief washeswith TBS, PH 7.4; and (vii) development according to the manufacturer'sinstructions, using an alkaline phosphatase substrate development kit(Novagen Inc.). Phage plaques which produced LBPs appeared asblue-colored “donuts” on the membranes.

The phage from Example 1 containing the LBP cDNAs were plaque-purifiedand converted into plasmid subclones by following a protocol called“Autosubcloning by Cre-mediated Plasmid Excision” provided by NovagenInc. DNA sequences were obtained by the dideoxynucleotidechain-termination method (Sanger et al., Proc. Natl. Acad. Sci., USA 74:5463-5467 (1977), and analyzed by an Applied Biosystems automatedsequencer. The open reading frame (ORF) of each cDNA was determined fromconsensus sequences obtained from both the sense and antisense strandsof the cDNAs. Sequencing confirmed that three previously unknown geneshad been isolated. Since the genes were selected by functional screeningfor LDL binding, the proteins coded by these genes were termed LDLbinding proteins (LBPs), specifically, LBP-1, LBP-2 and LBP-3. The cDNAsequences for rabbit LBP-1, LBP-2 and LBP-3 and the correspondingproteins are set forth in SEQ ID NOS: 10-14 and 48.

Based on their respective cDNA coding sequences, the sizes of therecombinant proteins were determined to be 16.2 kDa for LBP-1, 40 kDafor LBP-2, and 62.7 kDa for LBP-3.

Example 3 Northern Blot Analysis of Rabbit RNA Using LBP cDNA or cRNA

This example illustrates the size and tissue distribution of LBP mRNAs.Total RNA was isolated from different rabbit tissues: adrenals, thoracicaorta, abdominal aorta, ballooned and reendothelialized abdominal aorta,heart, kidney, smooth muscle cells, lung and liver, by Trizol reagent(Life Technologies) and concentrated by ethanol precipitation. Gelelectrophoresis of RNA was carried out in 1.2% agarose gel containing1×MOPS buffer (0.2M MOPS, pH 7.0; 50 mM sodium acetate; 5 mM EDTA, pH8.0) and 0.37M formaldehyde. Gels were loaded with 20 μg total RNA fromeach tissue examined and electrophoresed at 100 volts for 2 hr in 1×MOPSbuffer. RNAs were blotted onto supported nitrocellulose membranes(Schleicher & Schuell, Keene, N.H.) and immobilized by baking at 80° Cfor 2 hr. Hybridization to radiolabeled LBP-1, LBP-2 and LBP-3 cDNA orcRNA probes was carried out by standard procedures known to thoseskilled in the art (see, e.g., Ausubel et al., Current Protocols inMolecular Biology; John Wiley & Sons (1989)); signals were detected byautoradiography.

The results were as follows: the sizes of the mRNAs were about 1.3 kbfor LBP-1, about 2.3-2.5 kb for LBP-2, and about 4.7 kb for LBP-3.LBP-1, LBP-2 and LBP-3 mRNA were found in all tissues tested, but thehighest amount was in ballooned abdominal aorta.

Example 4 Isolation of Human LBP cDNAs and Genomic Clones

This example illustrates isolation of human LBP cDNAs. Human LBP cDNAclones were isolated from three cDNA libraries. A human fetal brain cDNAlibrary was obtained from Stratagene, LaJolla, Calif., a human liver anda human aorta cDNA library were obtained from Clontech, Palo Alto,Calif., and screened with a radiolabeled cDNA probe derived from rabbitLBP-1, LBP-2 or LBP-3, according to the method described in Law et al.,Gene Expression 4:77-84 (1994). Several strongly hybridizing clones wereidentified and plaque-purified. Clones were confirmed to be human LBP-1,LBP-2 and LBP-3, by DNA sequencing using the dideoxynucleotidechain-termination method and analysis by an Applied Biosystems automatedsequencer. The cDNA sequences and the corresponding proteins for humanLBP-1, LBP-2 and LBP-3 are set forth in SEQ ID NOS: 15, 16 and 17,respectively.

A human genomic library was screened with each of the LBP-1, LBP-2, andLBP-3 clones obtained from the cDNA library screening. Cloneshybridizing to each of the three cDNAs were isolated and sequenced. Thegenomic sequence for LBP-1, LBP-2, and LBP-3 are set forth in FIGS.22-24, respectively. The LBP-1 open reading frame spans four exons ofthe LBP-1 gene (FIG. 22; SEQ ID NO:49). The LBP-1 protein predicted bythe genomic sequence is identical to that predicted by the cDNA clonedescribed above. The LBP-2 open reading frame spans five exons of theLBP-2 gene (FIG. 23; SEQ ID NO:50). The LBP-2 protein predicted by thegenomic sequence differs from that predicted by the cDNA clone in thatit contains an additional 321 amino acids at its amino terminus (theLBP-2 cDNA is a 5′ truncation). The LBP-3 open reading frame spans tenexons of the LBP-3 gene (FIG. 24; SEQ ID NO:51). The LBP-3 proteinpredicted by the genomic sequence differs from that predicted by thecDNA clone in that it contains an additional 16 amino acids at its aminoterminus (the LBP-3 cDNA is a 5′ truncation) and an Asn at amino acidposition 130 (the cDNA predicts a Tyr at this position). A comparisonbetween the corresponding LBP-1, LBP-2 and LBP-3 protein sequences forrabbit and human are shown in FIGS. 19, 20 and 21.

Example 5 Isolation of Recombinant LBP-1. LBP-2 and LBP-3 RabbitProteins From E. coli

LBP cDNA was isolated from the original pEXlox plasmids obtained asdescribed in Examples 1 and 2, and subcloned into the pPRoEX-HT vector(Life Technologies) for recombinant protein expression. Induction of therecombinant protein by IPTG addition to transformed E. coli DH10Bcultures resulted in the expression of recombinant protein containing a6-histidine tag (N-terminal). This tagged protein was then purified fromwhole cell proteins by binding to Ni-NTA (nickel nitrilo-triacetic acid)as described in the protocol provided by the manufacturer (Qiagen, Inc.,Santa Clara, Calif.). The preparation obtained after the chromatographystep was approximately 90% pure; preparative SDS-PAGE was performed asthe final purification step.

When required by the characterization procedure, iodination of LBPs wascarried out using Iodobeads (Pierce, Rockford, Ill.). The lodobeads wereincubated with 500 μCi of Na¹²⁵I solution (17 Ci/mg) (New EnglandNuclear, Boston, Mass.) in a capped microfuge tube for 5 min at roomtemperature. The protein solution was added to the Iodobeads-Na¹²⁵Imicrofuge tube and incubated for 15 min at room temperature. At the endof this incubation, aliquots were removed for the determination of totalsoluble and TCA precipitable counts. The radiolabeled protein was thenprecipitated with cold acetone (2.5 vol; −20° C.; 2.5 hr). Followingthis incubation, precipitated protein was collected by centrifugation(14,000 g; 1 hr; room temperature) and resuspended in sample buffer (6 Murea/50 mM Tris, pH 8.0/2 mM EDTA). Integrity of the protein preparationwas assessed by SDS-PAGE.

The identities of the recombinant LBPs were confirmed using standardprotein sequencing protocols known to those skilled in the art. (APractical Guide for Protein and Peptide Purification forMicrosequencing, Matsudaira, ed., Academic Press, Inc., 2d edition(1993)). Analysis was performed using an Applied Biosystems Model 477AProtein Sequencer with on-line Model 120 PTH amino acid analyzer.

Example 6 Production of Antibodies to LBP-1, LBP-2 and LBP-3

This example illustrates the production of polyclonal antibodies toLBP-1, LBP-2 and LBP-3. A mixture of purified recombinant LBP protein(0.5 ml; 200 μg) and RIBI adjuvant (RIB1 ImmunoChem. Research, Inc.,Hamilton, Mont.) was injected subcutaneously into male guinea pigs(Dunkin Hartley; Hazelton Research Products, Inc., Denver, Pa.) at 3-5sites along the dorsal thoracic and abdominal regions of the guinea pig.Blood was collected by venipuncture on days 1 (pre-immune bleeding), 28,49 and 70. Booster injections were administered on days 21 (100 μg; SC),42 (50 μg; SC), and 63 (25 μg; SC). The titer of the guinea pigantiserum was evaluated by serial dilution “dot blotting.” Preimmuneantiserum was evaluated at the same time. After the third booster of LBPprotein, the titer against the recombinant protein reached a maximallevel with a detectable calorimetric response on a dot blot assay of 156pg.

Specificity of the polyclonal antibody for recombinant LBP-1, LBP-2 orLBP-3 was demonstrated using Western blot analysis. (Towbin et al.,Proc. Natl. Acad. Sci. USA 76: 4350 (1979)). The protein-antibodycomplex was visualized immunochemically with alkalinephosphatase-conjugated goat antiguinea pig IgG, followed by stainingwith nitro blue tetrazolium (BioRad Laboratories, Hercules, CA).Non-specific binding was blocked using 3% non-fat dry milk in Trisbuffered saline (100 mM Tris; 0.9% NaCl, pH 7.4).

Example 7 Immunohistochemical Characterization

This example illustrates the presence of LBPs in or on endothelial cellscovering plaques, in or on adjacent smooth muscle cells, and in theextracellular matrix. In addition, co-localization of LDL and LBPs wasdemonstrated. These results were obtained by examining ballooned rabbitarterial lesions and human atherosclerotic plaques byimmunohistochemical methods.

Ballooned deendothelialized aorta was obtained from rabbits which hadreceived a bolus injection of human LDL (3 mg; i.v.) 24 hr prior totissue collection. Human aortas containing atherosclerotic plaques wereobtained from routine autopsy specimens. Tissues were fixed in 10%buffered formalin (≦24 hr) and imbedded in paraffin using an automatedtissue-imbedding machine. Tissue sections were cut (5-7 μ) and mountedonto glass slides by incubating for 1 hr at 60° C. Sections weredeparaffinized. After a final wash with deionized H₂O, endogenousperoxidase activity was eliminated by incubating the sections with 1%H₂O₂/H₂O buffer for 5 min at room temperature. Sections were rinsed withphosphate buffered saline (PBS) for 5 min at room temperature andnonspecific binding was blocked with 5% normal goat serum or 5% normalrabbit serum depending on the source of the secondary antibody (Sigma,St. Louis, Mo.) (1 hr; room temperature). Sections were then incubatedwith a 1:50 dilution (in 5% normal goat serum/PBS) of a guinea pigpolyclonal antibody against the rabbit form of recombinant LBP-1, LBP-2or LBP-3. Controls included preimmune serum as well as specific antiserato LBP-1, LBP-2, or LBP-3 in which the primary antibody was completelyadsorbed and removed by incubation with recombinant LBP-1, LBP-2 orLBP-3 followed by centrifugation prior to incubation with the tissuesections. An affinity purified rabbit polyclonal antibody against humanapolipoprotein B (Polysciences Inc.; Warrington, Pa.) was used at adilution of 1:100 (in 5% normal rabbit serum/PBS). Sections wereincubated for 2 hr at room temperature in a humidified chamber. At theend of incubation, sections were rinsed with PBS and incubated with a1:200 dilution (in 5% normal goat serum/PBS) of goat anti-guinea pigbiotinylated IgG conjugate (Vector Laboratories, Burlingame, Calif.) ora 1:250 dilution (in 5% normal rabbit serum/PBS)of rabbit anti-goatbiotinylated IgG conjugate (Vector Laboratories, Burlingame, Calif.) for1 hr at room temperature in a humidified chamber. Sections were thenrinsed with PBS and antigen-antibody signal amplified usingavidin/biotin HRP conjugate (Vectastain ABC kit; Vector Laboratories,Burlingame, Calif.). Sections were developed using DAB substrate (4-6min; room temperature) and counterstained with hematoxylin. In theballooned rabbit artery, immunohistochemistry with the anti-LBP-1, LBP-2and LBP-3 antibodies showed that LBP-1, LBP-2 and LBP-3 were located inor on functionally modified endothelial cells at the edges ofregenerating endothelial islands, the same location in whichirreversible LDL binding has been demonstrated (Chang et al.,Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). LBP-1, LBP-2 andLBP-3 were also found in or on intimal smooth muscle cells underneaththe functionally modified endothelial cells, and to a lesser extent, inextracellular matrix. No LBP-1, LBP-2 or LBP-3 was detected in stilldeendothelialized areas, where LDL binding had been shown to bereversible (Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098(1992)). Immunohistochemistry of ballooned rabbit aorta with anti-humanapolipoprotein B antibodies showed the presence of LDL at the samelocations as that found for LBP-1, LBP-2 and LBP-3.

In the human atherosclerotic plaques taken at routine autopsies,immunohistochemistry with the anti-LBP-1,. anti-LBP-2 and anti-LBP-3antibodies showed that LBP-1, LBP-2, and LBP-3 were also found in or onendothelial cells covering plaques and in or on adjacent smooth musclecells. In the human tissue, there was greater evidence of LBP-1, LBP-2and LBP-3 in extracellular matrix.

The results obtained with paraffin sections were identical to those offrozen sections.

Example 8 Affinity Coelectrophoresis (ACE) Assays of LBPs and LDL or HDL

This example illustrates that binding occurs between LBP-1, LBP-2 orLBP-3 and LDL, and that this binding is specific, as illustrated by thefact that binding does not occur between LBP-1, LBP-2 or LBP-3 and HDL(high density lipoprotein). Analysis of the affinity and specificity ofrecombinant rabbit LBP-1, LBP-2 or LBP-3 binding to LDL was carried outusing the principle of affinity electrophoresis (Lee and Lander, Proc.Natl. Acad. Sci. USA 88:2768-2772 (1991)). Melted agarose (1%; 65° C.)was prepared in 50 mM sodium MOPS, pH 7.0; 125 mM sodium acetate, 0.5%CHAPS. A teflon comb consisting of nine parallel bars (45×4×4 mm/3 mmspacing between bars) was placed onto GelBond film (FMC Bioproducts,Rockland, ME) fitted to a plexiglass casting tray with the long axis ofthe bars parallel to the long axis of the casting tray. A teflon strip(66×1×1 mm) was placed on edge with the long axis parallel to the shortaxis of the casting tray, at a distance of 4 mm from the edge of theteflon comb. Melted agarose (>65° C.) was then poured to achieve aheight of approximately 4 mm. Removal of the comb and strip resulted ina gel containing nine 45×4×4 mm rectangular wells adjacent to a 66×1 mmslot. LDL or HDL samples were prepared in gel buffer (5 OmM sodium MOPS,pH 7.0, 125 mM sodium acetate) at twice the desired concentration.Samples were then mixed with an equal volume of melted agarose (in 50 mMMOPS, pH 7.0; 125 mM sodium acetate; 50° C.), pipetted into theappropriate rectangular wells and allowed to gel. The binding affinityand specificity of LBP-1 and LBP-3 was tested using severalconcentrations of LDL (540 to 14 nM) and HDL (2840177 nM). A constantamount (0.003 nM −0.016 nM) of ¹²⁵I-labeled LBP-1, LBP-2 or LBP-3(suspended in 50 mM sodium MOPS, pH 7.0; 125 mM sodium acetate; 0.5%bromphenol blue; 6% (wt/vol) sucrose) was loaded into the slot. Gelswere electrophoresed at 70v/2hr/20° C. At the end of the run, the gelswere air dried and retardation profiles were visualized by exposure ofX-ray films to the gels overnight at −70° C., with intensifying screens.

LDL retarded LBP-1, LBP-2 and LBP-3 migration through the gel in aconcentration-dependent, saturable manner, indicating that LBP-1, LBP-2and LBP-3 binding to LDL was highly specific. This conclusion issupported by the fact that HDL did not retard LBP-1, LBP-2 or LBP-3. Abinding curve generated from the affinity coelectrophoresis assayindicated that LBP-1 binds to LDL with a K_(d) of 25.6 nM, that LBP-2(rabbit clone 26) binds to LDL with a K_(d) of 100 nM, and that LBP-3(80 kDa fragment) binds to LDL with a K_(d) of 333 nM.

In addition to testing affinity and specificity of LBP-1, LBP-2 andLBP-3 binding to LDL, the ability of “cold” (i.e., non-radiolabeled)LBP-1, LBP-2 or LBP-3 to competitively inhibit radiolabeled LBP-1, LBP-2or LBP-3 binding to LDL, respectively, was tested. Competition studieswere carried out using fixed concentrations of cold LDL and radiolabeledLBP-1 and increasing amounts of cold recombinant LBP-1 (6-31 μM). TheACE assay samples and gel were prepared as described herein. Cold LBP-1inhibited binding of radiolabeled LBP-1 to LDL in aconcentration-dependent manner, cold LBP-2 inhibited binding ofradiolabeled LBP-2 to LDL in a concentration-dependent manner, and coldLBP-3 inhibited binding of radiolabeled LBP-3 to LDL in aconcentration-dependent manner.

Rabbit and human LBP-2 contain a long stretch of acidic amino acids atthe amino terminal (rabbit LBP-2 amino acid residues 338 through 365 andhuman LBP-2 amino acid residues 329 through 354). The possibility thatthis segment of LBP-2 was the LDL binding domain was tested bysubcloning two rabbit LBP-2 clones which differ from each other by thepresence or absence of this acidic region (clone 26 and clone 45,respectively) into expression vectors, by standard methods known tothose skilled in the art. ACE assays were then conducted in order toassess the affinity and specificity of the binding of these two clonesto LDL. LDL retarded clone 26 derived radiolabeled LBP-2 migrationthrough the gel in a concentration-dependent, saturable, manner whileclone 45 derived radiolabeled LBP-2 migration was not retarded.

Competition studies using fixed concentrations of cold LDL and clone 26derived radiolabeled LBP-2 and increasing concentrations of coldrecombinant LBP-2/clone 26 and LBP-a/clone 45 were carried out. Coldclone 26 derived LBP-2 inhibited binding of clone 26 derivedradiolabeled LBP-2 to LDL in a concentration-dependent manner. Clone 45derived LBP-2, on the other hand, did not affect the binding of clone 26derived radiolabeled LBP-2 to LDL. These results indicate that the longstretch of acidic amino acids contain a binding domain of LBP-2 to LDL.

Example 9 Affinity Coelectrophoreses (ACE) Assays of LBP-1 or LBP-2 andLDL in the Presence of Inhibitors

This example illustrates that binding between LBP-1 or LBP-2 and LDL isinhibited by polyglutamic acid or BHF-1. The ability of a third compoundto inhibit binding between two proteins previously shown to interact wastested by a modification of the ACE assays described in Example 8. Thethird compound was added to the top or wells together with theradiolabeled protein. If the third compound inhibited binding, theradiolabeled protein would run through the gel. If the third compounddid not inhibit binding, migration of the radiolabeled protein wasretarded by the protein cast into the gel.

Inhibition of LBP-1 /LDL or LBP-2/LDL binding by polyglutamic acid(average MW about 7500, corresponding to about 7 monomers) was shown bycasting a constant amount of LDL (148 nM) in all the rectangular lanes.A constant amount (1 μl) of ¹²⁵I-labeled LBP-1 or LBP-2 (0.003 nM −0.016nM) was loaded in the wells at the top of the gel, together withincreasing concentrations of polyglutamic acid (obtained from Sigma)(0-0.4 nM). The gel was electrophoresed at 70 volts for 2 hr, dried andplaced on X-ray film, with intensifying screens, overnight at −70° C.before the film was developed to determine the retardation profile ofLBP-1 and LBP-2. As the concentration of polyglutamic acid increased,retardation of radiolabeled LBP-1 and LBP-2 migration by LDL decreasedin a concentration-dependent manner, which showed that polyglutamic acidinhibited binding between LBP-1, LBP-2 and LDL.

Inhibition of LBP-1 /LDL binding by BHF-1 was shown by casting aconstant amount of LDL (148 nM) in all the rectangular lanes. A constantamount of ¹²⁵-labeled LBP-1 (0.003 nM −0.016 nM) was loaded in the wellsat the top of the gel, together with increasing concentrations ofBHF-1(0-10 nM), obtained as described in Example 15. The gel waselectrophoresed at 70 volts for 2 hr, dried and placed on X-ray film,with intensifying screens, overnight at −70° C. The film was thendeveloped to determine the retardation profile of ¹²⁵I-LBP-1. As theconcentration of BHF-1 increased, retardation of LBP-1 by LDL decreasedin a concentration-dependent manner, which demonstrated that BHF-1inhibited binding between LBP-1 and LDL.

Example 10 Affinity Coelectrophoreses (ACE) Assays for IdentifyingFragments, Analogs and Mimetics of LBPs which Bind to LDL

This example illustrates a method for identifying fragments, analogs ormimetics of LBPs which bind to LDL, and which thus can be used asinhibitors of LDL binding to LBP in the arterial walls, by occupyingbinding sites on LDL molecules, thereby rendering these sitesunavailable for binding to LBP in the arterial wall.

Fragments of LBPs are generated by chemical cleavage or synthesized fromthe known amino acid sequences. Samples of these fragments areindividually added (cold) to radiolabeled LBP as described in Example 8,to assess the inhibitory potency of the various fragments. By iterativeapplication of this procedure on progressively smaller portions offragments identified as inhibitory, the smallest active polypeptidefragment or fragments are identified. In a similar manner, analogs ofthe LBPs are tested to identify analogs which can act as inhibitors bybinding to LDL. And, similarly, mimetics of LBP (molecules whichresemble the conformation and/or charge distributions of the LDL-bindingsites on LBP molecules) are tested in a similar fashion to identifymolecules exhibiting affinities for the LDL-binding sites on LBP.

The affinities of the inhibitors so identified are at least as strong asthe affinity of LDL itself for the LDL-binding sites on LBP. Theinhibitors bind at least competitively, and some irreversibly andpreferentially as well, to the LDL-binding sites, thereby rendering suchsites unavailable for binding to humoral LDL.

Example 11 ELISA Assays

This example illustrates the use of ELISA plate assays for thequantification of a test compound's capacity to inhibit the binding ofLDL to a specific LBP.

In one example, the ELISA assay was carried out as follows: LDL wasdiluted in 50 mM Na₂HCO₃, pH 9.6/0.02% NaN₃ and added to the wells of a96-well plate (Immuno Ware 96-Well Reacti-Bind EIA Polystyrene Plates;Pierce (Rockford, Ill.)) to achieve a final concentration ranging from0.1 to 1 μg/well. The plates were incubated for 6 hr at roomtemperature. At the end of the incubation period, the wells were washed3 times with Tris-buffered saline, pH 7.4 (TBS), and blocked overnightwith 200 μl of 1% bovine serum albumin (BSA) in TBS/0.02% NaN₃ (Sigma;St. Louis Mo.) at room temperature. The wells were then incubated with200 μl of LBP protein (5-10 μg/well) in TBS and varying concentrationsof the test compound. Plates were incubated for 1 hr at roomtemperature. The wells were then washed three times with TBS and blockedfor 2 hr with 200 μl of 1% BSA in TBS/0.02% NaN₃ at room temperature. Atthe end of the incubation period, the wells were washed 3 times with TBSand a 1: 1000 dilution (in TBS/0.05% Tween 20) of the appropriate guineapig anti-LBP protein polyclonal antibody was added to the wells andincubated for 1 hr at room temperature. The wells were then washed 3times with TBS/0.05% Tween 20; a 1:30,000 dilution of goat anti-guineapig IgG alkaline phophatase conjugate (Sigma) was added to each well.Plates were incubated for 1 hr at room temperature. The wells werewashed 3 times with TBS/0.05% Tween 20 and a calorimetric reaction wascarried out by adding 200 ml of p-nitrophenyl phosphate substrate(Sigma; St. Louis Mo.) to the wells. The reaction was allowed to proceedfor 30 min at room temperature and stopped with 50 μl of 3N NaOH. Theabsorbance was determined at 405 nm using an ELISA plate reader. Thetest compound's effectiveness in blocking the binding of LDL to therecombinant protein was assessed by comparing the absorbance values ofcontrol and treated groups.

In a second example, the ELISA assay was carried out as follows: LDL wasdiluted in Tris-buffered saline, pH 7.4 (TBS) and added to the wells ofa 96-well plate (Immuno Ware 96-Well Reacti-Bind EIA Polystyrene Plates;Pierce (Rockford, Ill.)) to give a plate-saturating concentration of 0.2μg/well. The plate was incubated for 1 hr at room temperature, afterwhich the wells were washed three times with TBS, before being blockedfor 1 hr at room temperature with 1% bovine serum albumin (BSA in TBS).The wells were then washed twice with TBS before LBP-1 or LBP-2 (0.025μg/well), or LBP-3 (0.01 μg/well) were added, without and with varyingconcentrations of the test inhibitor compound. Each condition was set upin quadruplicate. The plate was incubated for 1 hr at room temperature,then washed three times with TBS/0.02% Tween 20 (TBS/Tween). Anappropriate dilution of guinea pig anti-LBP polyclonal antibody (1:750to 1:1500, depending on the antibody) was added to three wells for eachcondition and incubated for 1 hr. Anti-LBP antibody was replaced bybuffer for the fourth well of each condition, as a negative control.After 1 hr, the plate was again washed three times with TBS/Tween beforea 1: 10,000 dilution (in TBS/Tween) of goat anti-guinea pig IgG alkalinephosphatase-conjugated antibody (Sigma) was added to each well. Theplate was incubated for 1 hr at room temperature, then washed threetimes with TBS/Tween. A fresh solution of substrate was prepared from anAlkaline Phosphatase Substrate Kit (Bio-Rad, Hercules, Calif.) asfollows: Mix 1 ml 5× concentrated diethanolamine buffer with 4 mldistilled water. Add one tablet of p-nitrophenylphosphate (5 mg) andvortex until tablet is completely dissolved. Subtrate solution was addedto wells immediately. Increasing concentrations of diluted alkalinephosphatase-conjugated goat anti-guinea pig IgG (1:100,000 dilution inTBS/Tween) were added to five empty wells, followed by substrate, as apositive control. Following addition of substrate, the plate wasimmediately placed in an ELISA plate reader, allowed to stand at 37° C.,generally for 75 min, before absorbance was measured at 405 nm.Incubation in the ELISA reader at 37° C. was sometimes adjusted tooptimize absorbance (60-90 min). The effectiveness of the test inhibitorwas determined, after subtracting absorbance of negative controls, bycomparing absorbance in wells where an LBP was mixed with test inhibitorto absorbance in wells containing LBP with no inhibitor.

Alternatively, LBPs, rather than LDL, were bound to the plate.Recombinant LBP protein binding to LDL and the effect of varyingconcentration of the inhibitor on LBP-LDL binding was determined throughthe use of antibodies against LDL. This interaction was visualizedthrough the use of a secondary antibody conjugated to a reporter enzyme(e.g. alkaline phosphatase).

ELISA plate assays were used to screen agents which can affect thebinding of LBP proteins to LDL. For example, peptides derived from LBP-1and human LBP-3 protein sequences (BHF-1 and BHF-2, respectively) weresynthesized and have been shown to reduce the binding of LDL torecombinant LBP-1 and LBP-2 in this format. These results were inagreement with those obtained with the ACE assays.

Example 12 Administration of Humanized Antibodies Against LBPs so as toBlock LDL-Binding Sites on the LBPs

This example illustrates administration to patients of humanizedantibodies against LBP-1, LBP-2 or LBP-3 so as to block LDL-bindingsites on arterial LBP molecules. Mouse monoclonal antibodies arehumanized by recombinant DNA techniques and produced by standardprocedures known to those skilled in the art (Berkower, I., Curr. Opin.Biotechnol. 7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol.Lab 13: 26-28 (1995)) against LBPs and/or the LDL-binding sites on theLBPs. The corresponding Fab fragments are also produced, as described inGoding, J. W., Monoclonal Antibodies: Principles and Practice, AcademicPress, New York, N.Y. (1986). These antibodies are administeredparenterally in sufficient quantity so as to block LDL-binding sites onthe LBP molecules, i.e., 1-10 mg/kg daily. This prevents theirreversible arterial uptake of LDL that is required to facilitateoxidation of the LDL.

Example 13 Preparation of LDL

This example illustrates the preparation of LDL. LDL was prepared fromthe plasma of normolipemic donors (Chang et al., Arterioscler. Thromb.12:1088-1098 (1992)). 100 ml of whole blood was placed into tubescontaining 100 mM disodium EDTA. Plasma was separated from red bloodcells by low-speed centrifugation (2,000 g; 30 min; 4° C.). Plasmadensity was adjusted to 1.025 gm/ml with a solution of KBr andcentrifuged for 18-20 hr, 100,000×g, 12° C. Very low densitylipoproteins (VLDL) were removed from the tops of the centrifuge tubeswith a Pasteur pipette. The density of the infranate was raised to 1.050gm/ml with KBr solution and centrifuged for 22-24 hr, 100,000×g, 12° C.LDL was removed from the tops of the centrifuge tubes with a drawn outPasteur pipette tip. Purity of the LDL preparation was checked byOuchterlony double immunodiffusion against antibodies to human LDL,human HDL, human immunoglobulins, and human albumin. KBr was removedfrom the LDL solution by dialysis (1L,×2, approximately 16 hr) against0.9% saline, pH 9.0, containing 1 mM EDTA and 10 μM butylatedhydroxytoluene (BHT), the latter to prevent oxidation of LDL. Followingdialysis, LDL protein was measured by the method of Lowry (Lowry et al.,J. Biol. Chem. 193:265-275 (1951)), and the LDL was stored at 4° C.until use. LDL preparations were kept for no more than 4-6 weeks.

Example 14 Preparation of HDL

This example illustrates the preparation of HDL. HDL was prepared fromplasma of normolipemic donors. 100 ml of whole blood was placed intotubes containing 100 mM disodium EDTA and plasma was collected bycentrifugation (2000 g; 30 min; 4° C.). Apolipoprotein B containinglipoproteins present in plasma were then precipitated by the sequentialaddition of sodium heparin (5,000 units/ml) and MnCl₂ (1M) to achieve afinal concentration of 200 units/ml and 0.46 M, respectively (Warnickand Albers, J. Lipid Res. 19:65-76 (1978)). Samples were thencentrifuged, (2000 g; 1 hr; 4° C.). The supernatant was collected anddensity adjusted to 1.21 g/ml by the slow addition of solid KBr. HDL wasseparated by ultracentrifugation (100,000 g; >46 hr; 12° C.). Purity ofthe HDL preparation was assessed via Ouchterlony double immunodiffusiontest using antibodies against human HDL, human LDL, humanimmunoglobulins, and human albumin. HDL samples were dialyzed againstsaline pH 9.0/1 mM EDTA/10 μM BHT (4L; 24 hr/4° C.) and total proteinwas determined by the Lowry protein assay (Lowry et al., J. Biol. Chem.193:265-275 (1951)). HDL was stored at 4° C. until use. HDL preparationswere kept for no longer than 2 weeks.

Example 15 Synthesis of BHF-1

This example illustrates the synthesis of BHF-1, a fragment of human orrabbit LBP-1 which contains amino acid residues 14 through 33. BHF-1 wassynthesized using an Applied Biosystems Model 430A peptide synthesizerwith standard T-Boc NMP chemistry cycles. The sequence of BHF-1 is asfollows:

val-asp-val-asp-glu-tyr-asp-glu-asn-lys-phe-val-asp-glu-glu-asp-gly-gly-asp-gly(SEQ ID NO: 9).

After synthesis, the peptide was cleaved with hydrofluoric acid/anisole(10/l v/v) for 30 min at −10° C. and then incubated for 30 min at 0° C.BHF-1 was then precipitated and washed three times with cold diethylether. Amino acid coupling was monitored with the ninhydrin test (>99%).

The BHF-1 peptide was purified to homogeneity by high performance liquidchromatography on a reverse phase Vydac C₄ column (2.24×25 cm) using alinear gradient separation (2-98%B in 60 min) with a flow rate of 9ml/min. Buffer A consisted of 0.1 % trifluoroacetic acid (TFA)/Milli Qwater and Buffer B consisted of 0.085% TFA/80% acetonitrile. Thegradient was run at room temperature and absorbance monitored at 210 and277 nm.

Fast atom bombardment-mass spectrometry gave a protonated molecular ionpeak (M+H)⁺ at mn/z=2290.2, in good agreement with the calculated value.On amino acid analysis, experimental values for the relative abundanceof each amino acid in the peptide were in good-agreement withtheoretical values. The lyophilized peptide was stored at −20° C.

Example 16 In Vitro Screening for Agents Which Inhibit Binding BetweenLDL and LBPs

This example illustrates in vitro screening for agents which inhibitbinding between LDL and LBPs.

A candidate polypeptide for being an agent is chosen, e.g., LBP-1,LBP-2, LBP-3, BHF-1 or any other polypeptide. The shortest fragment ofthe polypeptide that inhibits LDL binding to LBPs in vitro isdetermined. Peptides are synthesized by standard techniques describedherein. Inhibition assays are performed using standard ELISA techniquesfor screening, and affinity coelectrophoresis (ACE) assays to confirmthe ELISA results, as described herein. Additional assays that can beused in this screening method include, e.g., fluorescence polarizationand pulsed ultra-filtration electrospray mass spectrometry. Shortpeptides ranging, e.g., from dimers to 20-mers are constructed acrosssequences of the candidate polypeptide whose chemical characteristicsmake them likely LDL binding sites, e.g., acidic regions. The ability ofshorter and shorter lengths of the peptides to inhibit LDL binding invitro and to mammalian cells in culture is tested. For example, theeffect of the peptide on inhibiting LDL binding in mammalian cellstransfected to express an LBP gene is tested. Each of the peptides soidentified as an inhibitor is tested with each of LBP-1, LBP-2 andLBP-3, to determine whether a single inhibitor works against all threeLBPs.

Once the minimum active sequence is determined, the peptide backbone ismodified so as to inhibit proteolysis, as discussed herein. For example,modification is accomplished by substitution of a sulfoxide for thecarbonyl, by reversing the peptide bond, by substituting a methylene forthe carbonyl group, or other similar standard methodology. See Spatola,A.F., “Peptide Backbone Modifications: A Structure-Activity Analysis ofPeptides Containing Amide Bond Surrogates, Conformational Constraints,and Related Backbone Replacements,” in Chemistry and Biochemistry ofAmino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein(ed.), Marcel Dekker, Inc., New York (1983). The ability of theseanalogs to inhibit LDL binding to the LBPs in vitro is tested in asimilar manner as for the natural peptides described above, e.g., byELISA, ACE, fluorescence polarization, and/or pulsed ultra-filtrationelectrospray mass spectrometry.

Example 17 In Vitro Screening With Cultured Mammalian Cells for AgentsWhich Inhibit Binding between LDL and LBPs

This example illustrates cell-based in vitro screening of agents whichhave been shown by in vitro tests such as ACE assay and ELISA to bepotential inhibitors of binding between LDL and LBPs.

Mammalian cells, such as 293 cells, which are commonly used forexpression of recombinant gene constructs, are used to develop celllines which express LBPs on the cell surface. This is done by subcloningLBP open reading frames (ORFS) into a mammalian expression plasmidvector, pDisplay (Invitrogen, Carlsbad, Calif.), which is designed toexpress the gene of interest on the cell surface. The use of mammaliancells to produce LBPs allows for their expression in a functionallyactive, native conformation. Therefore, stably transfected mammaliancell lines with surface expression of LBPs individually, or incombination, are particularly suitable for assaying and screeninginhibitors that block LDL binding in cell culture, as well as toevaluate the cytotoxicity of these compounds.

Specifically, LBP ORFs are amplified by PCR (Perkin Elmer, Foster City,Calif.) from cDNA templates using Taq polymerase (Perkin Elmer) andappropriate primers. The amplified LBP ORFs are purified by agarose gelelectrophoresis and extracted from gel slices with the Bio-Rad DNAPurification kit (Bio-Rad, Hercules, Calif.). The purified DNAs are thencut with the restriction enzymes Bgl II and Sal I (New England Biolabs,Beverly, Mass.) to generate cohesive ends, and purified again by agarosegel electrophoresis and DNA extraction as described above. The LBP ORFsare then subcloned into the Bgl II/Sal I sites in the mammalianexpression vector, pDisplay (Invitrogen) by ligation. Recombinantplasmids are established by transformation in E. coli strains TOP10(Invitrogen) or DH5α (Life Technologies, Grand Island, N.Y.).Recombinant pDisplay/LBP plasmid DNA is isolated from overnight E. colicultures with the Bio-Rad Plasmid Miniprep kit, cut with Bgl II/Sal I,and analyzed by agarose gel electrophoresis. LBP ORFs in successfullytransformed clones are verified by automated dideoxy DNA sequencing. Totransfect human kidney 293 cells, 1-2 μg of DNA is mixed with 6 μllipofectamine reagent (Life Technologies) and incubated with the cellsas described in the Life Technologies protocol. LBP expression intransfected cells is confirmed by Western blot analysis of cell extractsobtained 48 hr after transfection. To select for stably transfected 293cells, the antibiotic G418 (Life Technologies) is added to the growthmedium at a concentration of 800 μg/ml. Colonies resistant to G418 aretested for recombinant LBP expression by Western blot, and recombinantclones expressing LBPs are expanded, assayed for LDL binding and used totest compounds for their ability to inhibit LDL binding.

Example 18 In Vivo Screening for Agents Which Inhibit Binding BetweenLDL and LBPs

This example illustrates in vivo screening of agents which have beenshown by in vitro tests to be promising candidate inhibitors of bindingbetween LDL and LBPs.

In vivo inhibitory activity is first tested in the healingballoon-catheter deendothelialized rabbit aorta model of arterial injury(Roberts et al., J. Lipid Res. 24:1160-1167 (1983); Chang et al.,Arterioscler. Thromb. 12:1088-1098 (1992)). This model was shown to bean excellent analog for human atherosclerotic lesions. Other usefulanimal models for human atherosclerosis include Apo E knockout mice andLDL receptor knockout mice. Both of these mouse models are characterizedby high levels of plasma cholesterol and the development ofnaturally-occurring atherosclerotic-like lesions.

Each candidate inhibitor is tested in five to ten ballooned rabbits,while an equal number of rabbits receive a control peptide, or placebo.Four weeks following aortic deendothelialization, whenreendothelialization (healing) is partially complete, daily parenteral(intravenous or subcutaneous) or intragastric administration of thepeptides and the analogs begins at an initial concentration of 10 mg/kgbody weight, which is varied down, or up to 100 mg/kg depending onresults. 30 min later, a bolus of intravenously injected ₁₂₅I (or^(99m)Tc-) labeled LDL is given to test the candidate inhibitor'sability in short term studies to inhibit LDL sequestration in healingarterial lesions. If ¹²⁵I-LDL is used, the animals are sacrificed 8-24hr later, the aortas excised, washed and subjected to quantitativeautoradiography of excised aortas, as previously described (Roberts etal., J. Lipid Res. 24:1160-1167 (1983); Chang et al., Arterioscler.Thromb. 12:1088-1098 (1992)). If ^(99m)Tc-LDL is used, analysis is byexternal gamma camera imaging of the live anesthetized animal at 2-24hr, as previously described (Lees and Lees, Syndromes ofAtherosclerosis, in Fuster, ed., Futura Publishing Co., Armonk, N.Y.,pp. 385-401 (1996)), followed by sacrifice, excision and imaging of theexcised aorta. Immediately before the end of testing, the animals havestandard toxicity tests, including CBC, liver enzymes, and urinalysis.

The compounds which are most effective and least toxic are then testedin short term studies of rabbits fed a 2% cholesterol diet (Schwenke andCarew, Arteriosclerosis 9:895-907 (1989)). Each candidate inhibitor istested in five to ten rabbits, while an equal number of rabbits receivea control peptide, or placebo. Animals receive one or more doses per dayof the candidate inhibitor, or placebo, for up to two weeks. Dailyfrequency of doses is determined by route of administration. If activedrug or placebo are administered parenterally, they are given 1-3 timesdaily and the 2% cholesterol diet is continued. If drug or placebo aregiven orally, they are mixed with the 2% cholesterol diet. Schwenke andCarew (Arteriosclerosis 9:895-907 (1989)) have shown that the LDLconcentration in lesion-prone areas of the rabbit aorta is increased22-fold above normal in rabbits fed a 2% cholesterol diet for 16 days,and that the increased LDL content precedes the histological evidence ofearly atherosclerosis. Therefore, analysis of the effect of thecandidate inhibitors is tested two weeks after the start of cholesterolfeeding by injecting ¹²⁵I-LDL, allowing it to circulate for 8-24 hr, andthen performing quantitative autoradiography on the excised aortas ofboth test and control animals. If appropriate, quantitation of aorticcholesterol content is also carried out (Schwenke and Carew,Arteriosclerosis 9:895-907 (1989); Schwenke and Carew, Arteriosclerosis9:908-918 (1989).

The above procedures identify the most promising candidate inhibitors,as well as the best route and frequency of their administration.Inhibitors so identified are then tested in long-term studies ofcholesterol-fed rabbits. These tests are carried out in the same way asthe short-term cholesterol feeding studies, except that inhibitoreffectiveness is tested by injection of ¹²⁵I-LDL at longer intervalsfollowing the initiation of cholesterol feeding, and lesion-prone areasof the aorta are examined histologically for evidence ofatherosclerosis. Testing times are at two, four, and six months. Majorarteries are examined grossly and histologically or evidence and extentof atherosclerosis. If necessary, other accepted animal models, such asatherosclerosis-susceptible primates (Williams et al., Arterioscler.Thromb. Vast. Biol. 15:827-836 (1995)), genetically altered mice, and/orWatanabe rabbits are tested with short- and long-term cholesterolfeeding.

Example 19 In Vivo Inhibition of Radiolabeled LDL Accumulation in theBallooned Deendothelialized Rabbit Aorta via Induction of ActiveImmunity Against LBP Protein

This example illustrates the effect that induction of immunity againstLBP protein has on the accumulation of radiolabeled LDL in the ballooneddeendothelialized rabbit aorta model of atherosclerosis.

Immunity was induced in male New Zealand White rabbits (HazeltonResearch Products, Denver, PA) as follows: A mixture of purified humanrecombinant LBP-2 or BHF-1 peptide (1 ml; 1 mg) and RIBI adjuvant (RIBIImmunoChem Research, Inc., Hamilton, Mont.) was injected subcutaneouslyat 2-5 sites along the dorsal thoracic and abdominal regions of therabbits. Blood was collected by venipuncture on days 1 (preimmunebleeding), 35, 63, and 91. Booster injections were administered on days28 (500 μg; SC), 56 (250 μg; SC), and 84 (125 μg; SC).

The titer of the rabbits was evaluated by serial dilution using an ELISAplate format. Preimmune serum was evaluated at the same time. After thethird booster of LBP protein or peptide, the titer reached a maximallevel with a detectable calorimetric response on an ELISA plate of 156pg. Titer is defined as the maximum dilution of antibody which generatesan absorbance reading of 0.5 above control in 30 min. Specificity of thepolyclonal antibodies was demonstrated using Western blot analysis asdescribed in Example 6.

On day 93, the abdominal aorta of immunized and control rabbits wasdeendothelialized using a Fogarty number 4 embolectomy catheter (Changet al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). Four weeksafter ballooning, rabbits received a bolus injection of ¹²⁵I-labeled LDL(1 ml; i.v.). Blood samples were collected at 1 hr intervals for 8 hr,and 24 hr post injection. Blood samples were centrifuged for 30 min at2000 rpm (40° C.)and total activity present in the serum was determinedusing a Gamma counter. Total TCA precipitable counts were determined byaddition of TCA to the serum to a final concentration of 10% followed byincubation for 10 min at 4° C. Serum samples were then centrifuged (2000rpm; 30 min; 40° C.) and total activity present in the supernate wasdetermined. TCA precipitable counts were calculated by substration:total soluble counts minus counts present in the supernate after TCAprecipitation. Blood samples for the determination of antibody titerswere collected prior to the injection of the radiolabeled LDL.

After 24 hr, the rabbits were injected intravenously with 5% Evan's bluedye which was allowed to circulate for 15 min. Areas of the aorta inwhich the endothelial covering is absent stain blue while those areascovered by endothelium remain unstained. At the end of the incubationperiod, the rabbits were euthanized and the abdominal and thoracic aortawere dissected out, rinsed, and fixed overnight in 10% TCA at roomtemperature. The aortas were then rinsed exhaustively with physiologicalsaline, weighed, counted, blotted dry and placed onto X-ray film inorder to visualize the pattern of radiolabeled LDL accumulation in thedeendothelialized rabbit abdominal aorta.

Immunization of rabbits against recombinant human LBP-2 or BHF-1 peptidealtered the pattern of radiolabeled LDL accumulation in the ballooneddeendothelialized abdominal aorta. When corrected for dosage, andpercent reendothelialization, immunized-ballooned rabbits had loweraccumulation of radiolabeled LDL compared to nonimmune-balloonedrabbits. These results indicate that active immunization against LBPprovides an effective means by which the accumulation of LDL in theinjured arterial wall can be modified.

Example 20 Screening Agents in Humans Which Inhibit Binding Between LDLand LBPs

Human studies are carried out according to standard FDA protocols fortesting of new drugs for safety (Phase I), efficacy (Phase II), andefficacy compared to other treatments-(Phase III). Subjects, who areenrolled into studies after giving informed consent, are between theages of 18 and 70. Women who are pregnant, or likely to become pregnant,or subjects with diseases other than primary atherosclerosis, such ascancer, liver disease, or diabetes, are excluded. Subjects selected forstudy in FDA Phase II and Phase III trials have atherosclerotic diseasepreviously documented by standard techniques, such as ultrasound and/orangiography, or are known to be at high risk of atherosclerosis byvirtue of having at least one first degree relative with documentedatherosclerosis. Subjects themselves have normal or abnormal plasmalipids. Initial testing includes 20-50 subjects on active drug and 20-50subjects, matched for age, sex, and atherosclerotic status, on placebo.The number of subjects is pre-determined by the number needed forstatistical significance. Endpoints for inhibitor efficacy includesultrasound measurements of carotid artery thickness in high risksubjects, as well as in subjects with known carotid or coronary disease;atherosclerotic events; atherosclerotic deaths; and all-cause deaths inall subjects. Non-invasive analysis (carotid artery thickness byultrasound) as per Stadler (Med. and Biol. 22:25-34 (1996)) are carriedout at 6- to 12-month intervals for 3 years. Atherosclerotic events anddeaths, as well as all-cause deaths are tabulated at 3 years.

Oral dosage of drug in FDA Phase I trials ranges from 0.01 to 10 gm/day,and is determined by results of animal studies, extrapolated on a per kgbasis. Based on data obtained from Phase I studies, the dose range andfrequency are narrowed in Phase II and III trials. If parenteraladministration of drug is determined by animal studies to be the onlyeffective method, parenteral administration in human subjects is testedby injection, as well as by the transdermal and nasal insufflationroutes. Testing of parenteral drug follows the same outline as that fororal administration.

The optimal treatment schedule and dosage for humans is thusestablished.

Example 21 Treating an Individual Having Atherosclerosis With BHF-1

This example illustrates a method for treating an individual havingatherosclerosis with an LBP fragment, e.g., BHF-1, so as to decrease thelevels of arterially bound LDL in the individual. BHF-1 is obtained asdescribed herein. The BHF-1 is administered to the mammal intravenouslyas a bolus or as an injection at a concentration of 0.5-10 mg/kg bodyweight. Such administrations are repeated indefinitely in order toprevent the development or progression of symptomatic atherosclerosis,just as is done currently with cholesterol lowering drugs. Stablesubjects are examined twice yearly to evaluate the extent of anyatherosclerotic disease by physical exam and non-invasive studies, suchas carotid artery thickness, ultrasound, and/or gamma camera imaging ofthe major arteries, to determine if atherosclerotic lesions are present,and, if previously present, have regressed or progressed. Such a regimenresults in treatment of the atherosclerosis.

Example 22 In Vivo Reduction of Atherosclerosis in Apo E Knockout Miceby Immunization With LBPs

Separate immunization experiments were performed with each of LBP-1,LBP-2, and LBP-3. Immunity was induced by injecting apo E knockout micewith the LBP protein (LBP-1, LBP-2, or LBP-3) together with an RIBIadjuvant (RIBI ImmunoChem Research, Inc., Hamilton, Mont.). Apo Eknockout mice (Jackson Laboratories, Bar Harbor, Me.) are hyperlipidemicand thus a model for human atherosclerosis. Apo E knockout mice havehigh levels of plasma cholesterol and develop naturally-occurringatherosclerotic-like lesions.

Four week old apo E knockout mice (Jackson Laboratories, Bar Harbor,Me.) were ear tagged, randomly assigned to different cages and weighed.Body weights were determined weekly. Animals were allowed to habituatefor 1 week. Normal rodent chow was provided ad libitum and animals weremaintained in a 12:12 light:dark cycle. The following four groups ofmice were treated with either recombinant LBP proteins (40 pg ofrecombinant protein/mouse) plus RIBI adjuvant or RIBI adjuvant alone(control group).

LBP-1: Immunized with rabbit recombinant LBP-1 (6-His tag).

LBP-2: Immunized with rabbit recombinant LBP-2 clone 26 (6-His tag).

LBP-3: Immunized with rabbit recombinant LBP-3 (6-His tag).

Control: Received adjuvant.

Blood samples (pre-immune serum) were collected prior to the initialinjection of recombinant protein and RIBI adjuvant (as described in themanufacturer's manual). After 21 days, mice received a booster injection(half-initial dose) and were then bled seven days later. Titer wasdefined as the maximum dilution of serum that yielded a change inabsorbance equivalent to 2× that of control serum (60 min; 37° C.). Theamount of recombinant protein per well was 100 ng.

Booster injections took place at 21 day intervals until an average titervalue of 1:10,000 was reached. At this time, mice were switched towestern type diet (Harland Teklad, Madison, Wis.) and fed ad libitum.Blood samples were collected at this time (retro-orbital sinus bleedingtechnique) and monthly thereafter.

Blood samples were analyzed for total cholesterol, HDL cholesterol, andtriglyceride concentration with a commercially available totalcholesterol and triglycerides assay kits (Sigma; St. Louis Mo.) using anELISA format. HDL concentration was determined after Apo B containinglipoproteins were precipitated using heparin/MnCl₂.

Apo E knockout mice were sacrificed at 26 weeks of age. The mice wereanesthetized with methoxyfluorane and exanguinated via cardiac puncture.A midline thoracotomy was performed, a cannula inserted into the rightventricle and perfusate allowed unrestricted flow via an incision intothe right atrium. The mice were perfused with saline, followed by 10%phosphate buffered formalin until fasciculations stopped. At this time,the aorta was exposed and adventitial fat removed in situ. The aorta wasthen removed from the heart down to the iliac bifurcation and placed in10% phosphate buffered formalin overnight.

The aorta was stained as follows: after a brief 70% ethanol rinse, itwas immersed in a filtered solution of 0.5% (weight/volume) Sudan IV in35% ethanol/50% acetone with continuous shaking for 10 minutes at roomtemperature. Unbound dye was removed by incubating the aorta in an 80%ethanol solution with shaking until the background color was clear. Thevessel was then rinsed in distilled water, placed in physiologicalsaline and opened longitudinally from the aortic arch down to the iliacbifurcation. The vessel was pinned out and photographed. Photographswere then digitized using an Astra 1200S scanner (UMAX TechnologiesInc., Freemont, Calif.) and a commercially available graphics program(Canvas; Deneba Software, Miami Fla.). Total and lesion areas weredetermined using the signal processing toolbox of MATLAB (The MathworksInc., Natick, Mass.). Percent involvement was calculated by dividinglesion area by total area.

A second analysis was done to measure aortic atherosclerosis by acholesterol extraction method whereby cholesterol is determined as aunit weight of artery. This method may be more accurate in measuringlesion size than attempting to measure the thickness of many sections.Specifically, the weight of an artery was measured, then the cholesterolwas extracted. Aortic cholesterol content was then measured bygas-liquid chromatography. The amount of cholesterol per unit weight ofartery was then determined.

After the first booster injection, some of the apo E knockout miceimmunized against LBP-1 had relatively high anti-LBP-1 titers (>1:5000)while others in the same group exhibited moderate levels (<1:500 to<1:1000). LBP-2/26 titers were low in the apoE knockout mice (<1:500) atthis time. LBP-3 titers ranged from moderate to low (>1:500 to <1:1000)to low (<1:500) in the apoE knockout mice.

After the second booster injection, Apo E knockout mice immunizedagainst LBP-1 had moderate to high titers (>1:1000 to <1:8000). Apo Eknockout mice immunized against LBP-2/26 had moderate titer levels(>1:2000). LBP-3 titers range from moderate to high (>1:1000 to >1:8000)in the Apo knockout mice.

After the third booster injection, most of the mice immunized againstLBP-1 had relatively high titers (>1:10,000) while others had moderateto high titers (>1000 to <1:10,000). Some of the Apo E knockout mice hadmoderate (<1:5000) to low (<1:1000) titers. LBP-3 titers ranged fromhigh (>1:5000 to ≦1:10,000) to moderate (>1:1000 to <1:5000).

Data were analyzed using T-tests and Wilcoxons. Immunization againstLBP-1, LBP-2/26 or LBP-3 did not have a significant effect (P>0.05) onbody weight of Apo E knockout mice. Due to the small sample size and thelarge variability present in the Apo E knockout mice, it was notpossible to determine whether immunization against LBP-1, LBP-2/26 orLBP-3 had an effect on total cholesterol, HDL cholesterol ortriglycerides concentration, but it did not appear to.

Immunization against LBP-1 or LBP-3 did not have a significant effect(P>0.05) on lesions of the apo E knockout mice or LDL receptor negativeknockout mice. However, immunization of the apo E knockout mice againstLBP-2 had a significant effect on lesion area (Table 2), and, onceoutliers were deleted, a significant effect on arterial wall cholesterolcontent (Table 3). The LBP-2 immunized apo E knockout mice hadsignificantly reduced aortic atherosclerosis as compared to the control,non-immunized mice. Without being bound to any particular theory, thecirculating antibodies generated against LBP-2 proteins are thought toblock LDL binding to the artery wall.

TABLE 2 Lesion Area in LBP-Immunized Apo E Mice Apo E Lesion AreaTreated Area P-Value Mice % Coverage Change Wilcoxon Control 9.40 LBP-16.05 −0.36% 0.07 LBP-2 6.01 −0.36% 0.01 LBP-3 7.14 −0.24% 0.36

TABLE 3 Arterial Cholesterol Content in LBP-Immunized Apo E MiceArterial Wall Treated Area Apo E Cholesterol (ug Cholesterol P-ValueMice cholesterol/mg aorta) Change Wilcoxon Control 6.33 LBP-1 3.82−0.40% 0.14 LBP-2 3.28 −0.48% 0.07 LBP-2 (outliers 1.83 −0.71% 0.01deleted) LBP-3 4.48 −0.29% 0.20

Example 23 In Vivo Reduction of Atherosclerosis in LDL Receptor KnockoutMice by Immunization With BHF-1

An immunization experiment was performed with the BHF-1 peptide. LDLreceptor (LDLR) knockout mice (B6,129S-Ldlr^(tmlHer), JacksonLaboratories, Bar Harbor, Me.) were injected with the BHF-1 peptide (seeExample 15 for methods of synthesizing the BHF-1 peptide) together withan RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, Mont.). LDLRknockout mice are hyperlipidemic and thus a model for humanatherosclerosis. LDLR knockout mice have high levels of plasmacholesterol and develop naturally-occurring atherosclerotic-likelesions.

Four week old LDLR knockout mice were ear tagged, randomly assigned todifferent cages and weighed. Body weights were subsequently determinedweekly. Animals were allowed to habituate for one week prior toexperimentation. Normal rodent chow was provided ad libitum and animalswere maintained in a 12:12 light:dark cycle. Animals were divided intoexperimental and control groups, as follows: (1) experimental, 16 micewere immunized with the BHF-1.20.L peptide; (2) control, 8 mice wereimmunized against bovine serum albumin.

Mice in the experimental group received subcutaneous injections (9.99μg/g body weight; 200 μl final volume) of the BHF-1.20.L peptide dailyfor 2 weeks, from 5 to 7 weeks of age, prior to the initial injectionwith the peptide and adjuvant. Blood samples (pre-immune serum) werecollected prior to the initial injection of BHF-1.20.L and RIBI adjuvant(50 μg of peptide/mouse) (as described in the manufacturer's manual) at7 weeks of age. After 21 days, mice received a booster injection(half-initial dose) and were then bled 7 days later. Titer was definedas the maximum dilution of serum that yielded a change in absorbanceequivalent to 2× that of control serum (60 min; 37° C.). The amount ofpeptide per well was 100 ng. Booster injections took place at 21 daysinterval.

Blood samples were analyzed for total cholesterol, HDL cholesterol, andtriglyceride concentration, using commercially available totalcholesterol and triglycerides assay kits (Sigma; St. Louis Mo.) (ELISA).HDL concentration was determined after Apo B containing lipoproteinswere precipitated using heparin/MnCl₂.

When fed a normal rodent chow, total serum cholesterol concentration inLDLR knockout mice remains relatively low. A high fat diet, on the otherhand, results in an increase in total serum cholesterol concentration inthese mice. The animals were thus switched at 16 weeks of age to amodified “Western Type” diet (0.1% cholesterol content) (Harland Teklad,Madison, Wis.) and fed ad libitum. This diet was expected to increasethe total serum cholesterol concentration to a range of 600-800 mg/dl,thereby increasing the rate of lesion formation. Blood samples werecollected at 18 weeks of age (retro-orbital sinus bleeding technique)and monthly thereafter.

At 30 weeks of age, the mice were sacrificed and aorta were removed asdescribed in Example 22. Aortic atherosclerosis was measured by thecholesterol extraction method described in Example 22, wherebycholesterol is determined as a unit weight of artery.

Immunization against BHF-1.20.L had no effect on body weight of LDLRknockout mice. Consumption of the modified “Western Type” diet for 12weeks significantly (P<.05) increased total serum cholesterol, HDLcholesterol and triglycerides in both experimental and control animals.Levels of total serum cholesterol, HDL serum cholesterol and serumtriglyceride concentration were not significantly different (P>0.05)between experimental and control animals.

Mice that were immunized with the BHF-1 peptide had 24% less aorticcholesterol content (P>0.037) as compared to the control, non-immunizedmice. Without being bound to any particular theory, the immunization isthought to generate circulating antibodies against the BHF-1 peptide.These antibodies are thought to block LDL binding to the artery wall,thereby reducing aortic cholesterol content.

Those skilled in the art will be able to ascertain using no more thanroutine experimentation, many equivalents of the specific embodiments ofthe invention described herein. These and all other equivalents areintended to be encompassed by the following claims.

53 1 151 PRT Oryctolagus cuniculus 1 Met Ser Lys Asn Thr Val Ser Ser AlaArg Phe Arg Lys Val Asp Val 1 5 10 15 Asp Glu Tyr Asp Glu Asn Lys PheVal Asp Glu Glu Asp Gly Gly Asp 20 25 30 Gly Gln Ala Gly Pro Asp Glu GlyGlu Val Asp Ser Cys Leu Arg Gln 35 40 45 Gly Asn Met Thr Ala Ala Leu GlnAla Ala Leu Lys Asn Pro Pro Ile 50 55 60 Asn Thr Arg Ser Gln Ala Val LysAsp Arg Ala Gly Ser Ile Val Leu 65 70 75 80 Lys Val Leu Ile Ser Phe LysAla Gly Asp Ile Glu Lys Ala Val Gln 85 90 95 Ser Leu Asp Arg Asn Gly ValAsp Leu Leu Met Lys Tyr Ile Tyr Lys 100 105 110 Gly Phe Glu Ser Pro SerAsp Asn Ser Ser Ala Val Leu Leu Gln Trp 115 120 125 His Glu Lys Ala LeuAla Ala Gly Gly Val Gly Ser Ile Val Arg Val 130 135 140 Leu Thr Ala ArgLys Thr Val 145 150 2 317 PRT Oryctolagus cuniculus VARIANT (1)...(317)Xaa = Any Amino Acid 2 Asp Cys Arg Ser Ser Ser Asn Asn Arg Xaa Pro LysGly Gly Ala Ala 1 5 10 15 Arg Ala Gly Gly Pro Ala Arg Pro Val Ser LeuArg Glu Val Val Arg 20 25 30 Tyr Leu Gly Gly Ser Ser Gly Ala Gly Gly ArgLeu Thr Arg Gly Arg 35 40 45 Val Gln Gly Leu Leu Glu Glu Glu Ala Ala AlaArg Gly Arg Leu Glu 50 55 60 Arg Thr Arg Leu Gly Ala Leu Ala Leu Pro ArgGly Asp Arg Pro Gly 65 70 75 80 Arg Ala Pro Pro Ala Ala Ser Ala Arg AlaAla Arg Asn Lys Arg Ala 85 90 95 Gly Glu Glu Arg Val Leu Glu Lys Glu GluGlu Glu Glu Glu Glu Glu 100 105 110 Asp Asp Glu Asp Asp Asp Asp Asp ValVal Ser Glu Gly Ser Glu Val 115 120 125 Pro Glu Ser Asp Arg Pro Ala GlyAla Gln His His Gln Leu Asn Gly 130 135 140 Gly Glu Arg Gly Pro Gln ThrAla Lys Glu Arg Ala Lys Glu Trp Ser 145 150 155 160 Leu Cys Gly Pro HisPro Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala 165 170 175 Gly Ser Gly ThrArg Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu 180 185 190 Gly Gly SerAla Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val 195 200 205 Pro LeuPro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro 210 215 220 PheGly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala Asp Pro Val Glu 225 230 235240 Trp Thr Val Met Asp Val Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro 245250 255 Glu Gln Ala Thr Ala Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu260 265 270 Leu Leu Met Gln Arg Thr Asp Val Leu Thr Gly Leu Ser Ile ArgLeu 275 280 285 Gly Pro Ala Leu Lys Ile Tyr Glu His His Ile Lys Val LeuGln Gln 290 295 300 Gly His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly305 310 315 3 232 PRT Oryctolagus cuniculus 3 Ala Ser Ala Arg Ala AlaArg Asn Lys Arg Ala Gly Glu Glu Arg Val 1 5 10 15 Leu Glu Lys Glu GluGlu Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp 20 25 30 Asp Asp Asp Val ValSer Glu Gly Ser Glu Val Pro Glu Ser Asp Arg 35 40 45 Pro Ala Gly Ala GlnHis His Gln Leu Asn Gly Gly Glu Arg Gly Pro 50 55 60 Gln Thr Ala Lys GluArg Ala Lys Glu Trp Ser Leu Cys Gly Pro His 65 70 75 80 Pro Gly Gln GluGlu Gly Arg Gly Pro Ala Ala Gly Ser Gly Thr Arg 85 90 95 Gln Val Phe SerMet Ala Ala Leu Ser Lys Glu Gly Gly Ser Ala Ser 100 105 110 Ser Thr ThrGly Pro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro Gly 115 120 125 Lys ProAla Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro Ala 130 135 140 GlyArg Lys Glu Lys Pro Ala Asp Pro Val Glu Trp Thr Val Met Asp 145 150 155160 Val Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr Ala 165170 175 Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln Arg180 185 190 Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala LeuLys 195 200 205 Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His PheGlu Asp 210 215 220 Asp Asp Pro Glu Gly Phe Leu Gly 225 230 4 252 PRTOryctolagus cuniculus 4 Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly AspArg Pro Gly Arg 1 5 10 15 Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala ArgAsn Lys Arg Ala Gly 20 25 30 Glu Glu Arg Val Leu Glu Lys Glu Glu Glu GluGlu Glu Glu Glu Asp 35 40 45 Asp Glu Asp Asp Asp Asp Asp Val Val Ser GluGly Ser Glu Val Pro 50 55 60 Glu Ser Asp Arg Pro Ala Gly Ala Gln His HisGln Leu Asn Gly Gly 65 70 75 80 Glu Arg Gly Pro Gln Thr Ala Lys Glu ArgAla Lys Glu Trp Ser Leu 85 90 95 Cys Gly Pro His Pro Gly Gln Glu Glu GlyArg Gly Pro Ala Ala Gly 100 105 110 Ser Gly Thr Arg Gln Val Phe Ser MetAla Ala Leu Ser Lys Glu Gly 115 120 125 Gly Ser Ala Ser Ser Thr Thr GlyPro Asp Ser Pro Ser Pro Val Pro 130 135 140 Leu Pro Pro Gly Lys Pro AlaLeu Pro Gly Ala Asp Gly Thr Pro Phe 145 150 155 160 Gly Cys Pro Ala GlyArg Lys Glu Lys Pro Ala Asp Pro Val Glu Trp 165 170 175 Thr Val Met AspVal Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu 180 185 190 Gln Ala ThrAla Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu 195 200 205 Leu MetGln Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly 210 215 220 ProAla Leu Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly 225 230 235240 His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly 245 250 5 557 PRTOryctolagus cuniculus 5 Met Lys Asn Gln Asp Lys Lys Asn Gly Ala Ala LysGln Pro Asn Pro 1 5 10 15 Lys Ser Ser Pro Gly Gln Pro Glu Ala Gly AlaGlu Gly Ala Gln Gly 20 25 30 Arg Pro Gly Arg Pro Ala Pro Ala Arg Glu AlaGlu Gly Ala Ser Ser 35 40 45 Gln Ala Pro Gly Arg Pro Glu Gly Ala Gln AlaLys Thr Ala Gln Pro 50 55 60 Gly Ala Leu Cys Asp Val Ser Glu Glu Leu SerArg Gln Leu Glu Asp 65 70 75 80 Ile Leu Ser Thr Tyr Cys Val Asp Asn AsnGln Gly Ala Pro Gly Glu 85 90 95 Asp Gly Val Gln Gly Glu Pro Pro Glu ProGlu Asp Ala Glu Lys Ser 100 105 110 Arg Ala Tyr Val Ala Arg Asn Gly GluPro Glu Pro Gly Thr Pro Val 115 120 125 Val Asn Gly Glu Lys Glu Thr SerLys Ala Glu Pro Gly Thr Glu Glu 130 135 140 Ile Arg Thr Ser Asp Glu ValGly Asp Arg Asp His Arg Arg Pro Gln 145 150 155 160 Glu Lys Lys Lys AlaLys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170 175 Gln Thr Leu AsnThr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 180 185 190 Cys Lys LysTyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln Lys 195 200 205 Gln MetLys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu Lys 210 215 220 AspHis Leu Arg Gly Glu His Ser Lys Ala Ile Leu Ala Arg Ser Lys 225 230 235240 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn Arg Ser Leu Lys 245250 255 Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu Glu Lys Arg Lys Glu260 265 270 Val Thr Ser His Phe Gln Met Thr Leu Asn Asp Ile Gln Leu GlnMet 275 280 285 Glu Gln His Asn Glu Arg Asn Ser Lys Leu Arg Gln Glu AsnMet Glu 290 295 300 Leu Ala Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr GluLeu Arg Glu 305 310 315 320 Glu His Ile Asp Lys Val Phe Lys His Lys AspLeu Gln Gln Gln Leu 325 330 335 Val Asp Ala Lys Leu Gln Gln Ala Gln GluMet Leu Lys Glu Ala Glu 340 345 350 Glu Arg His Gln Arg Glu Lys Asp PheLeu Leu Lys Glu Ala Val Glu 355 360 365 Ser Gln Arg Met Cys Glu Leu MetLys Gln Gln Glu Thr His Leu Lys 370 375 380 Gln Gln Leu Ala Leu Tyr ThrGlu Lys Phe Glu Glu Phe Gln Asn Thr 385 390 395 400 Leu Ser Lys Ser SerGlu Val Phe Thr Thr Phe Lys Gln Glu Met Glu 405 410 415 Lys Met Thr LysLys Ile Lys Lys Leu Glu Lys Glu Thr Thr Met Tyr 420 425 430 Arg Ser ArgTrp Glu Ser Ser Asn Lys Ala Leu Leu Glu Met Ala Glu 435 440 445 Glu LysThr Leu Arg Asp Lys Glu Leu Glu Gly Leu Gln Val Lys Ile 450 455 460 GlnArg Leu Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465 470 475480 Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly Pro Val 485490 495 Ser Asp Ser Gly Pro Glu Arg Arg Pro Glu Pro Ala Thr Thr Ser Lys500 505 510 Glu Gln Gly Val Glu Gly Pro Gly Ala Gln Val Pro Asn Ser ProArg 515 520 525 Ala Thr Asp Ala Ser Cys Cys Ala Gly Ala Pro Ser Thr GluAla Ser 530 535 540 Gly Gln Thr Gly Pro Gln Glu Pro Thr Thr Ala Thr Ala545 550 555 6 151 PRT Homo sapiens 6 Met Ser Lys Asn Thr Val Ser Ser AlaArg Phe Arg Lys Val Asp Val 1 5 10 15 Asp Glu Tyr Asp Glu Asn Lys PheVal Asp Glu Glu Asp Gly Gly Asp 20 25 30 Gly Gln Ala Gly Pro Asp Glu GlyGlu Val Asp Ser Cys Leu Arg Gln 35 40 45 Gly Asn Met Thr Ala Ala Leu GlnAla Ala Leu Lys Asn Pro Pro Ile 50 55 60 Asn Thr Lys Ser Gln Ala Val LysAsp Arg Ala Gly Ser Ile Val Leu 65 70 75 80 Lys Val Leu Ile Ser Phe LysAla Asn Asp Ile Glu Lys Ala Val Gln 85 90 95 Ser Leu Asp Lys Asn Gly ValAsp Leu Leu Met Lys Tyr Ile Tyr Lys 100 105 110 Gly Phe Glu Ser Pro SerAsp Asn Ser Ser Ala Met Leu Leu Gln Trp 115 120 125 His Glu Lys Ala LeuAla Ala Gly Gly Val Gly Ser Ile Val Arg Val 130 135 140 Leu Thr Ala ArgLys Thr Val 145 150 7 217 PRT Homo sapiens 7 Glu Glu Arg Val Leu Glu LysGlu Glu Glu Glu Asp Asp Asp Glu Asp 1 5 10 15 Glu Asp Glu Glu Asp AspVal Ser Glu Gly Ser Glu Val Pro Glu Ser 20 25 30 Asp Arg Pro Ala Gly AlaGln His His Gln Leu Asn Gly Glu Arg Gly 35 40 45 Pro Gln Ser Ala Lys GluArg Val Lys Glu Trp Thr Pro Cys Gly Pro 50 55 60 His Gln Gly Gln Asp GluGly Arg Gly Pro Ala Pro Gly Ser Gly Thr 65 70 75 80 Arg Gln Val Phe SerMet Ala Ala Met Asn Lys Glu Gly Gly Thr Ala 85 90 95 Ser Val Ala Thr GlyPro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro 100 105 110 Gly Lys Pro AlaLeu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro 115 120 125 Pro Gly ArgLys Glu Lys Pro Ser Asp Pro Val Glu Trp Thr Val Met 130 135 140 Asp ValVal Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr 145 150 155 160Ala Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln 165 170175 Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala Leu 180185 190 Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His Phe Glu195 200 205 Asp Asp Asp Pro Asp Gly Phe Leu Gly 210 215 8 530 PRT Homosapiens 8 Lys Ser Ser Pro Gly Gln Pro Glu Ala Gly Pro Glu Gly Ala GlnGlu 1 5 10 15 Arg Pro Ser Gln Ala Ala Pro Ala Val Glu Ala Glu Gly ProGly Ser 20 25 30 Ser Gln Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala Arg ThrAla Gln 35 40 45 Ser Gly Ala Leu Arg Asp Val Ser Glu Glu Leu Ser Arg GlnLeu Glu 50 55 60 Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly GlyPro Gly 65 70 75 80 Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu AspAla Glu Lys 85 90 95 Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu Pro Glu ProThr Pro Val 100 105 110 Val Tyr Gly Glu Lys Glu Pro Ser Lys Gly Asp ProAsn Thr Glu Glu 115 120 125 Ile Arg Gln Ser Asp Glu Val Gly Asp Arg AspHis Arg Arg Pro Gln 130 135 140 Glu Lys Lys Lys Ala Lys Gly Leu Gly LysGlu Ile Thr Leu Leu Met 145 150 155 160 Gln Thr Leu Asn Thr Leu Ser ThrPro Glu Glu Lys Leu Ala Ala Leu 165 170 175 Cys Lys Lys Tyr Ala Glu LeuLeu Glu Glu His Arg Asn Ser Gln Lys 180 185 190 Gln Met Lys Leu Leu GlnLys Lys Gln Ser Gln Leu Val Gln Glu Lys 195 200 205 Asp His Leu Arg GlyGlu His Ser Lys Ala Val Leu Ala Arg Ser Lys 210 215 220 Leu Glu Ser LeuCys Arg Glu Leu Gln Arg His Asn Arg Ser Leu Lys 225 230 235 240 Glu GluGly Val Gln Arg Ala Arg Glu Glu Glu Glu Lys Arg Lys Glu 245 250 255 ValThr Ser His Phe Gln Val Thr Leu Asn Asp Ile Gln Leu Gln Met 260 265 270Glu Gln His Asn Glu Arg Asn Ser Lys Leu Arg Gln Glu Asn Met Glu 275 280285 Leu Ala Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 290295 300 Glu His Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu305 310 315 320 Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys GluAla Glu 325 330 335 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys GluAla Val Glu 340 345 350 Ser Gln Arg Met Cys Glu Leu Met Lys Gln Gln GluThr His Leu Lys 355 360 365 Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe GluGlu Phe Gln Asn Thr 370 375 380 Leu Ser Lys Ser Ser Glu Val Phe Thr ThrPhe Lys Gln Glu Met Glu 385 390 395 400 Lys Met Thr Lys Lys Ile Lys LysLeu Glu Lys Glu Thr Thr Met Tyr 405 410 415 Arg Ser Arg Trp Glu Ser SerAsn Lys Ala Leu Leu Glu Met Ala Glu 420 425 430 Glu Lys Thr Val Arg AspLys Glu Leu Glu Gly Leu Gln Val Lys Ile 435 440 445 Gln Arg Leu Glu LysLeu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 450 455 460 Leu Asn Lys ArgVal Gln Asp Leu Ser Ala Gly Gly Gln Gly Ser Leu 465 470 475 480 Thr AspSer Gly Pro Glu Arg Arg Pro Glu Gly Pro Gly Ala Gln Ala 485 490 495 ProSer Ser Pro Arg Val Thr Glu Ala Pro Cys Tyr Pro Gly Ala Pro 500 505 510Ser Thr Glu Ala Ser Gly Gln Thr Gly Pro Gln Glu Pro Thr Ser Ala 515 520525 Arg Ala 530 9 20 PRT Homo sapiens 9 Val Asp Val Asp Glu Tyr Asp GluAsn Lys Phe Val Asp Glu Glu Asp 1 5 10 15 Gly Gly Asp Gly 20 10 1404 DNAOryctolagus cuniculus CDS (58)...(510) 10 aagcctcgca gcggtcggggcggcgccgcg gaggctcgag ggcggcgggc ggcggcg atg 60 Met 1 tcg aag aac acggtg tcg tcg gcg cgg ttc cgg aag gtg gac gtg gat 108 Ser Lys Asn Thr ValSer Ser Ala Arg Phe Arg Lys Val Asp Val Asp 5 10 15 gag tac gac gag aacaag ttc gtg gac gag gaa gac ggc ggc gac ggc 156 Glu Tyr Asp Glu Asn LysPhe Val Asp Glu Glu Asp Gly Gly Asp Gly 20 25 30 cag gcg ggg ccg gac gagggc gag gtg gac tcg tgc ctg cgg caa ggg 204 Gln Ala Gly Pro Asp Glu GlyGlu Val Asp Ser Cys Leu Arg Gln Gly 35 40 45 aac atg aca gcc gcc ctg caggcg gcg ctg aag aac cct ccc atc aac 252 Asn Met Thr Ala Ala Leu Gln AlaAla Leu Lys Asn Pro Pro Ile Asn 50 55 60 65 acc agg agc cag gcg gtg aaggac cgg gca ggc agc atc gtg ctg aag 300 Thr Arg Ser Gln Ala Val Lys AspArg Ala Gly Ser Ile Val Leu Lys 70 75 80 gtg ctc atc tcc ttc aag gcc ggcgac ata gaa aag gcc gtg cag tcc 348 Val Leu Ile Ser Phe Lys Ala Gly AspIle Glu Lys Ala Val Gln Ser 85 90 95 ctg gac agg aac ggc gtg gac ctg ctcatg aag tac atc tac aag ggc 396 Leu Asp Arg Asn Gly Val Asp Leu Leu MetLys Tyr Ile Tyr Lys Gly 100 105 110 ttc gag agc ccc tcc gac aac agc agcgcc gtg ctc ctg cag tgg cac 444 Phe Glu Ser Pro Ser Asp Asn Ser Ser AlaVal Leu Leu Gln Trp His 115 120 125 gag aag gcg ctg gct gca gga gga gtgggc tcc atc gtc cgt gtc ctg 492 Glu Lys Ala Leu Ala Ala Gly Gly Val GlySer Ile Val Arg Val Leu 130 135 140 145 act gca agg aaa acc gtgtagcctggca ggaacgggtg cctgccgggg 540 Thr Ala Arg Lys Thr Val 150agcgggagct gccggtacaa agaccaaaac gcccagatgc cgccgctgcc ctgtgggcgg 600cgtctgttcc cagcttcgct ttttcccttt cccgtgtctg tcaggattac ataaggtttc 660ccttcgtgag aatcggagtg gcgcagaggg tcctgttcat acgcgccgtg cgtccggctg 720tgtaagaccc ctgccttcag tgtccttgag caacggtagc gtgtcgccgg ctgggtttgg 780ttttgtcgtg gagggatctg gtcagaattt gaggccagtt tcctaactca ttgctggtca 840ggaaatgatc ttcatttaaa aaaaaaaaaa agactggcag ctattatgca aaactggacc 900ctcttccctt atttaagcag agtgagtttc tggaaccagt ggtgcccccc cccccgcccc 960ggccgccgtc ctgctcaagg gaagcctccc tgcagagcag cagagcccct gggcaggagc 1020gccgcgtccc gctcccagga gacagcatgc gcggtcacgc ggcacttcct gtgcctccca 1080gccccagtgc cccggagttc ttcagggcga cagggacctc agaagactgg atccgatcca 1140gacagacgcc cattcttggt tcagctcagt gttttcaaaa ggaacgtgct accgtgggta 1200gagcacactg gttctcagaa cacggccggc gcttgacggt tgtcacagct ccagaacaaa 1260tcctgggaga caggcgagcg cgagtcgccg ggcaggaatt ccacacactc gtgctgtttt 1320tgatacctgc tttttgtttt gttttgtaaa aatgatgcac ttgagaaaat aaaacgtcag 1380tgttgacaaa aaaaaaaaaa aaaa 1404 11 1617 DNA Oryctolagus cuniculus CDS(1)...(951) 11 gac tgc cgc agc agc agc aac aac cgc tag ccg aag ggt ggcgcg gcg 48 Asp Cys Arg Ser Ser Ser Asn Asn Arg * Pro Lys Gly Gly Ala Ala1 5 10 15 cgg gcc ggc ggc ccg gcg cgg ccc gtg agc ctg cgg gaa gtc gtgcgc 96 Arg Ala Gly Gly Pro Ala Arg Pro Val Ser Leu Arg Glu Val Val Arg20 25 30 tac ctc ggg ggt agc agc ggc gct ggc ggc cgc ctg acc cgc ggc cgc144 Tyr Leu Gly Gly Ser Ser Gly Ala Gly Gly Arg Leu Thr Arg Gly Arg 3540 45 gtg cag ggt ctg ctg gaa gag gag gcg gcg gcg cgg ggc cgc ctg gag192 Val Gln Gly Leu Leu Glu Glu Glu Ala Ala Ala Arg Gly Arg Leu Glu 5055 60 cgc acc cgt ctc gga gcg ctt gcg ctg ccc cgc ggg gac agg ccc gga240 Arg Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly Asp Arg Pro Gly 6570 75 cgg gcg cca ccg gcc gcc agc gcc cgc gcg gcg cgg aac aag aga gct288 Arg Ala Pro Pro Ala Ala Ser Ala Arg Ala Ala Arg Asn Lys Arg Ala 8085 90 95 ggc gag gag cga gtg ctt gaa aag gag gag gag gag gag gag gag gaa336 Gly Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Glu Glu Glu Glu 100105 110 gac gac gag gac gac gac gac gac gtc gtg tcc gag ggc tcg gag gtg384 Asp Asp Glu Asp Asp Asp Asp Asp Val Val Ser Glu Gly Ser Glu Val 115120 125 ccc gag agc gat cgt ccc gcg ggt gcg cag cat cac cag ctg aat ggc432 Pro Glu Ser Asp Arg Pro Ala Gly Ala Gln His His Gln Leu Asn Gly 130135 140 ggc gag cgc ggc ccg cag acc gcc aag gag cgg gcc aag gag tgg tcg480 Gly Glu Arg Gly Pro Gln Thr Ala Lys Glu Arg Ala Lys Glu Trp Ser 145150 155 ctg tgt ggc ccc cac cct ggc cag gag gaa ggg cgg ggg ccg gcc gcg528 Leu Cys Gly Pro His Pro Gly Gln Glu Glu Gly Arg Gly Pro Ala Ala 160165 170 175 ggc agt ggc acc cgc cag gtg ttc tcc atg gcg gcc ttg agt aaggag 576 Gly Ser Gly Thr Arg Gln Val Phe Ser Met Ala Ala Leu Ser Lys Glu180 185 190 ggg gga tca gcc tct tcg acc acc ggg cct gac tcc ccg tcc ccggtg 624 Gly Gly Ser Ala Ser Ser Thr Thr Gly Pro Asp Ser Pro Ser Pro Val195 200 205 cct ttg ccc ccc ggg aag cca gcc ctc cca gga gcc gat ggg accccc 672 Pro Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro210 215 220 ttt ggc tgc cct gcc ggg cgc aaa gag aag ccg gca gac ccc gtggag 720 Phe Gly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala Asp Pro Val Glu225 230 235 tgg aca gtc atg gac gtc gtg gag tac ttc acc gag gcg ggc ttccct 768 Trp Thr Val Met Asp Val Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro240 245 250 255 gag caa gcc acg gct ttc cag gag cag gag atc gac ggc aagtcc ctg 816 Glu Gln Ala Thr Ala Phe Gln Glu Gln Glu Ile Asp Gly Lys SerLeu 260 265 270 ctg ctc atg cag cgc acc gat gtc ctc acc ggc ctg tcc atccgc ctg 864 Leu Leu Met Gln Arg Thr Asp Val Leu Thr Gly Leu Ser Ile ArgLeu 275 280 285 ggg cca gcg ttg aaa atc tat gag cac cat atc aag gtg ctgcag cag 912 Gly Pro Ala Leu Lys Ile Tyr Glu His His Ile Lys Val Leu GlnGln 290 295 300 ggt cac ttc gag gac gat gac ccg gaa ggc ttc ctg ggatgagcacaga 961 Gly His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly 305310 315 gccgccgcgc cccttgtccc cacccccacc ccgcctggac ccattcctgcctccatgtca 1021 cccaaggtgt cccagaggcc aggagctgga ctgggcaggc gaggggtgcggacctaccct 1081 gattctggta gggggcgggg ccttgctgtg ctcattgcta cccccccaccccgtgtgtgt 1141 ctctgcacct gcccccagca cacccctccc ggagcctgga tgtcgcctgggactctggcc 1201 tgctcatttt gcccccagat cagccccctc cctccctcct gtcccaggacattttttaaa 1261 agaaaaaaag gaaaaaaaaa aattggggag ggggctggga aggtgccccaagatcctcct 1321 cggcccaacc aggtgtttat tcctatatat atatatatat gttttgttctgcctgttttt 1381 cgttttttgg tgcgtggcct ttcttccctc ccaccaccac tcatggccccagccctgctc 1441 gccctgtcgg cgggagcagc tgggaatggg aggagggtgg gaccttgggtctgtctccca 1501 ccctctctcc cgttggttct gttgtcgctc cagctggctg tattgctttttaatattgca 1561 ccgaagggtt gttttttttt ttttaaataa aattttaaaa aaaggaaaaaaaaaaa 1617 12 1362 DNA Oryctolagus cuniculus CDS (1)...(696) 12 gcc agcgcc cgc gcg gcg cgg aac aag aga gct ggc gag gag cga gtg 48 Ala Ser AlaArg Ala Ala Arg Asn Lys Arg Ala Gly Glu Glu Arg Val 1 5 10 15 ctt gaaaag gag gag gag gag gag gag gag gaa gac gac gag gac gac 96 Leu Glu LysGlu Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp 20 25 30 gac gac gacgtc gtg tcc gag ggc tcg gag gtg ccc gag agc gat cgt 144 Asp Asp Asp ValVal Ser Glu Gly Ser Glu Val Pro Glu Ser Asp Arg 35 40 45 ccc gcg ggt gcgcag cat cac cag ctg aat ggc ggc gag cgc ggc ccg 192 Pro Ala Gly Ala GlnHis His Gln Leu Asn Gly Gly Glu Arg Gly Pro 50 55 60 cag acc gcc aag gagcgg gcc aag gag tgg tcg ctg tgt ggc ccc cac 240 Gln Thr Ala Lys Glu ArgAla Lys Glu Trp Ser Leu Cys Gly Pro His 65 70 75 80 cct ggc cag gag gaaggg cgg ggg ccg gcc gcg ggc agt ggc acc cgc 288 Pro Gly Gln Glu Glu GlyArg Gly Pro Ala Ala Gly Ser Gly Thr Arg 85 90 95 cag gtg ttc tcc atg gcggcc ttg agt aag gag ggg gga tca gcc tct 336 Gln Val Phe Ser Met Ala AlaLeu Ser Lys Glu Gly Gly Ser Ala Ser 100 105 110 tcg acc acc ggg cct gactcc ccg tcc ccg gtg cct ttg ccc ccc ggg 384 Ser Thr Thr Gly Pro Asp SerPro Ser Pro Val Pro Leu Pro Pro Gly 115 120 125 aag cca gcc ctc cca ggagcc gat ggg acc ccc ttt ggc tgc cct gcc 432 Lys Pro Ala Leu Pro Gly AlaAsp Gly Thr Pro Phe Gly Cys Pro Ala 130 135 140 ggg cgc aaa gag aag ccggca gac ccc gtg gag tgg aca gtc atg gac 480 Gly Arg Lys Glu Lys Pro AlaAsp Pro Val Glu Trp Thr Val Met Asp 145 150 155 160 gtc gtg gag tac ttcacc gag gcg ggc ttc cct gag caa gcc acg gct 528 Val Val Glu Tyr Phe ThrGlu Ala Gly Phe Pro Glu Gln Ala Thr Ala 165 170 175 ttc cag gag cag gagatc gac ggc aag tcc ctg ctg ctc atg cag cgc 576 Phe Gln Glu Gln Glu IleAsp Gly Lys Ser Leu Leu Leu Met Gln Arg 180 185 190 acc gat gtc ctc accggc ctg tcc atc cgc ctg ggg cca gcg ttg aaa 624 Thr Asp Val Leu Thr GlyLeu Ser Ile Arg Leu Gly Pro Ala Leu Lys 195 200 205 atc tat gag cac catatc aag gtg ctg cag cag ggt cac ttc gag gac 672 Ile Tyr Glu His His IleLys Val Leu Gln Gln Gly His Phe Glu Asp 210 215 220 gat gac ccg gaa ggcttc ctg gga tgagcacaga gccgccgcgc cccttgtccc 726 Asp Asp Pro Glu Gly PheLeu Gly 225 230 cacccccacc ccgcctggac ccattcctgc ctccatgtca cccaaggtgtcccagaggcc 786 aggagctgga ctgggcaggc gaggggtgcg gacctaccct gattctggtagggggcgggg 846 ccttgctgtg ctcattgcta cccccccacc ccgtgtgtgt ctctgcacctgcccccagca 906 cacccctccc ggagcctgga tgtcgcctgg gactctggcc tgctcattttgcccccagat 966 cagccccctc cctccctcct gtcccaggac attttttaaa agaaaaaaaggaaaaaaaaa 1026 aattggggag ggggctggga aggtgcccca agatcctcct cggcccaaccaggtgtttat 1086 tcctatatat atatatatat gttttgttct gcctgttttt cgttttttggtgcgtggcct 1146 ttcttccctc ccaccaccac tcatggcccc agccctgctc gccctgtcggcgggagcagc 1206 tgggaatggg aggagggtgg gaccttgggt ctgtctccca ccctctctcccgttggttct 1266 gttgtcgctc cagctggctg tattgctttt taatattgca ccgaagggttgttttttttt 1326 ttttaaataa aattttaaaa aaaggaaaaa aaaaaa 1362 13 1422 DNAOryctolagus cuniculus CDS (1)...(756) 13 acc cgt ctc gga gcg ctt gcg ctgccc cgc ggg gac agg ccc gga cgg 48 Thr Arg Leu Gly Ala Leu Ala Leu ProArg Gly Asp Arg Pro Gly Arg 1 5 10 15 gcg cca ccg gcc gcc agc gcc cgcgcg gcg cgg aac aag aga gct ggc 96 Ala Pro Pro Ala Ala Ser Ala Arg AlaAla Arg Asn Lys Arg Ala Gly 20 25 30 gag gag cga gtg ctt gaa aag gag gaggag gag gag gag gag gaa gac 144 Glu Glu Arg Val Leu Glu Lys Glu Glu GluGlu Glu Glu Glu Glu Asp 35 40 45 gac gag gac gac gac gac gac gtc gtg tccgag ggc tcg gag gtg ccc 192 Asp Glu Asp Asp Asp Asp Asp Val Val Ser GluGly Ser Glu Val Pro 50 55 60 gag agc gat cgt ccc gcg ggt gcg cag cat caccag ctg aat ggc ggc 240 Glu Ser Asp Arg Pro Ala Gly Ala Gln His His GlnLeu Asn Gly Gly 65 70 75 80 gag cgc ggc ccg cag acc gcc aag gag cgg gccaag gag tgg tcg ctg 288 Glu Arg Gly Pro Gln Thr Ala Lys Glu Arg Ala LysGlu Trp Ser Leu 85 90 95 tgt ggc ccc cac cct ggc cag gag gaa ggg cgg gggccg gcc gcg ggc 336 Cys Gly Pro His Pro Gly Gln Glu Glu Gly Arg Gly ProAla Ala Gly 100 105 110 agt ggc acc cgc cag gtg ttc tcc atg gcg gcc ttgagt aag gag ggg 384 Ser Gly Thr Arg Gln Val Phe Ser Met Ala Ala Leu SerLys Glu Gly 115 120 125 gga tca gcc tct tcg acc acc ggg cct gac tcc ccgtcc ccg gtg cct 432 Gly Ser Ala Ser Ser Thr Thr Gly Pro Asp Ser Pro SerPro Val Pro 130 135 140 ttg ccc ccc ggg aag cca gcc ctc cca gga gcc gatggg acc ccc ttt 480 Leu Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp GlyThr Pro Phe 145 150 155 160 ggc tgc cct gcc ggg cgc aaa gag aag ccg gcagac ccc gtg gag tgg 528 Gly Cys Pro Ala Gly Arg Lys Glu Lys Pro Ala AspPro Val Glu Trp 165 170 175 aca gtc atg gac gtc gtg gag tac ttc acc gaggcg ggc ttc cct gag 576 Thr Val Met Asp Val Val Glu Tyr Phe Thr Glu AlaGly Phe Pro Glu 180 185 190 caa gcc acg gct ttc cag gag cag gag atc gacggc aag tcc ctg ctg 624 Gln Ala Thr Ala Phe Gln Glu Gln Glu Ile Asp GlyLys Ser Leu Leu 195 200 205 ctc atg cag cgc acc gat gtc ctc acc ggc ctgtcc atc cgc ctg ggg 672 Leu Met Gln Arg Thr Asp Val Leu Thr Gly Leu SerIle Arg Leu Gly 210 215 220 cca gcg ttg aaa atc tat gag cac cat atc aaggtg ctg cag cag ggt 720 Pro Ala Leu Lys Ile Tyr Glu His His Ile Lys ValLeu Gln Gln Gly 225 230 235 240 cac ttc gag gac gat gac ccg gaa ggc ttcctg gga tgagcacaga 766 His Phe Glu Asp Asp Asp Pro Glu Gly Phe Leu Gly245 250 gccgccgcgc cccttgtccc cacccccacc ccgcctggac ccattcctgcctccatgtca 826 cccaaggtgt cccagaggcc aggagctgga ctgggcaggc gaggggtgcggacctaccct 886 gattctggta gggggcgggg ccttgctgtg ctcattgcta cccccccaccccgtgtgtgt 946 ctctgcacct gcccccagca cacccctccc ggagcctgga tgtcgcctgggactctggcc 1006 tgctcatttt gcccccagat cagccccctc cctccctcct gtcccaggacattttttaaa 1066 agaaaaaaag gaaaaaaaaa aattggggag ggggctggga aggtgccccaagatcctcct 1126 cggcccaacc aggtgtttat tcctatatat atatatatat gttttgttctgcctgttttt 1186 cgttttttgg tgcgtggcct ttcttccctc ccaccaccac tcatggccccagccctgctc 1246 gccctgtcgg cgggagcagc tgggaatggg aggagggtgg gaccttgggtctgtctccca 1306 ccctctctcc cgttggttct gttgtcgctc cagctggctg tattgctttttaatattgca 1366 ccgaagggtt gttttttttt ttttaaataa aattttaaaa aaaggaaaaaaaaaaa 1422 14 4722 DNA Oryctolagus cuniculus CDS (61)...(1731) 14gtggaaaata gcaactgtgt ttctcaagga tccaatccca acctaaggtg gcagcgcaca 60 atgaag aat caa gac aaa aag aac ggg gct gcc aaa cag ccc aac ccc 108 Met LysAsn Gln Asp Lys Lys Asn Gly Ala Ala Lys Gln Pro Asn Pro 1 5 10 15 aaaagc agc ccg gga cag ccg gaa gca gga gcg gag gga gcc cag ggg 156 Lys SerSer Pro Gly Gln Pro Glu Ala Gly Ala Glu Gly Ala Gln Gly 20 25 30 cgg cccggc cgg ccg gcc ccc gcc cga gaa gcc gaa ggt gcc agc agc 204 Arg Pro GlyArg Pro Ala Pro Ala Arg Glu Ala Glu Gly Ala Ser Ser 35 40 45 cag gct cccggg agg ccg gag ggg gct caa gcc aaa act gct cag cct 252 Gln Ala Pro GlyArg Pro Glu Gly Ala Gln Ala Lys Thr Ala Gln Pro 50 55 60 ggg gcg ctc tgtgat gtc tct gag gag ctg agc cgc cag ttg gaa gac 300 Gly Ala Leu Cys AspVal Ser Glu Glu Leu Ser Arg Gln Leu Glu Asp 65 70 75 80 ata ctc agt acatac tgt gtg gac aac aac cag ggg gcc ccg ggt gag 348 Ile Leu Ser Thr TyrCys Val Asp Asn Asn Gln Gly Ala Pro Gly Glu 85 90 95 gat ggg gtc cag ggtgag ccc cct gaa cct gaa gat gca gag aag tct 396 Asp Gly Val Gln Gly GluPro Pro Glu Pro Glu Asp Ala Glu Lys Ser 100 105 110 cgc gcc tat gtg gcaagg aat ggg gag ccg gag ccg ggc acc cca gta 444 Arg Ala Tyr Val Ala ArgAsn Gly Glu Pro Glu Pro Gly Thr Pro Val 115 120 125 gtc aat ggc gag aaggag acc tcc aag gca gag ccg ggc acg gaa gag 492 Val Asn Gly Glu Lys GluThr Ser Lys Ala Glu Pro Gly Thr Glu Glu 130 135 140 atc cgg acg agc gatgag gtc gga gac cga gac cac cgg agg cca cag 540 Ile Arg Thr Ser Asp GluVal Gly Asp Arg Asp His Arg Arg Pro Gln 145 150 155 160 gaa aag aag aaggcc aag ggt ctg gga aag gag atc acg ctg ctg atg 588 Glu Lys Lys Lys AlaLys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met 165 170 175 cag aca ctg aacacg ctg agc acc cca gag gag aag ctg gcg gct ctg 636 Gln Thr Leu Asn ThrLeu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu 180 185 190 tgc aag aag tatgcg gaa ctg ctc gag gag cac cgg aac tcg cag aag 684 Cys Lys Lys Tyr AlaGlu Leu Leu Glu Glu His Arg Asn Ser Gln Lys 195 200 205 cag atg aag ctgctg cag aag aag cag agc cag ctg gtg cag gag aag 732 Gln Met Lys Leu LeuGln Lys Lys Gln Ser Gln Leu Val Gln Glu Lys 210 215 220 gac cac ctg cgtggc gag cac agc aag gcc atc ctg gcc cgc agc aag 780 Asp His Leu Arg GlyGlu His Ser Lys Ala Ile Leu Ala Arg Ser Lys 225 230 235 240 ctc gag agcctg tgc cgg gag ctg cag cgg cac aac cgc tcg ctc aag 828 Leu Glu Ser LeuCys Arg Glu Leu Gln Arg His Asn Arg Ser Leu Lys 245 250 255 gaa gaa ggtgtg cag cga gcc cga gag gag gag gag aag cgc aag gag 876 Glu Glu Gly ValGln Arg Ala Arg Glu Glu Glu Glu Lys Arg Lys Glu 260 265 270 gtg acg tcacac ttc cag atg acg ctc aac gac att cag ctg cag atg 924 Val Thr Ser HisPhe Gln Met Thr Leu Asn Asp Ile Gln Leu Gln Met 275 280 285 gag cag cacaac gag cgc aac tcc aag ctg cgc cag gag aac atg gag 972 Glu Gln His AsnGlu Arg Asn Ser Lys Leu Arg Gln Glu Asn Met Glu 290 295 300 ctg gcc gagcgg ctc aag aag ctg att gag cag tac gag ctg cga gaa 1020 Leu Ala Glu ArgLeu Lys Lys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310 315 320 gag cacatc gac aaa gtc ttc aaa cac aag gat ctg cag cag cag ctg 1068 Glu His IleAsp Lys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 325 330 335 gtg gacgcc aag ctc cag cag gcc cag gag atg ctg aag gag gca gag 1116 Val Asp AlaLys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala Glu 340 345 350 gag cggcac cag cgg gag aag gac ttt ctc ctg aag gag gcc gtg gag 1164 Glu Arg HisGln Arg Glu Lys Asp Phe Leu Leu Lys Glu Ala Val Glu 355 360 365 tcc cagagg atg tgc gag ctg atg aag caa cag gag acc cac ctg aag 1212 Ser Gln ArgMet Cys Glu Leu Met Lys Gln Gln Glu Thr His Leu Lys 370 375 380 cag cagctt gcc cta tac aca gag aag ttt gag gag ttc cag aac act 1260 Gln Gln LeuAla Leu Tyr Thr Glu Lys Phe Glu Glu Phe Gln Asn Thr 385 390 395 400 ctttcc aaa agc agc gag gtg ttc acc aca ttc aaa cag gaa atg gaa 1308 Leu SerLys Ser Ser Glu Val Phe Thr Thr Phe Lys Gln Glu Met Glu 405 410 415 aagatg aca aag aag atc aag aag ctg gag aaa gag acc acc atg tac 1356 Lys MetThr Lys Lys Ile Lys Lys Leu Glu Lys Glu Thr Thr Met Tyr 420 425 430 cgttcc cgg tgg gag agc agc aac aag gcc ctg ctt gag atg gct gag 1404 Arg SerArg Trp Glu Ser Ser Asn Lys Ala Leu Leu Glu Met Ala Glu 435 440 445 gagaaa aca ctc cgg gac aaa gag ctg gaa ggc ctg cag gtg aaa atc 1452 Glu LysThr Leu Arg Asp Lys Glu Leu Glu Gly Leu Gln Val Lys Ile 450 455 460 cagcgg ctg gag aag ctg tgc cgg gca ctg cag aca gag cgc aat gac 1500 Gln ArgLeu Glu Lys Leu Cys Arg Ala Leu Gln Thr Glu Arg Asn Asp 465 470 475 480ctg aac aag agg gtg cag gac ctg agt gcc ggt ggc cag ggc ccc gtc 1548 LeuAsn Lys Arg Val Gln Asp Leu Ser Ala Gly Gly Gln Gly Pro Val 485 490 495tcc gac agc ggt cct gag cgg agg cca gag ccc gcc acc acc tcc aag 1596 SerAsp Ser Gly Pro Glu Arg Arg Pro Glu Pro Ala Thr Thr Ser Lys 500 505 510gag cag ggt gtc gag ggc ccc ggg gct caa gta ccc aac tct cca agg 1644 GluGln Gly Val Glu Gly Pro Gly Ala Gln Val Pro Asn Ser Pro Arg 515 520 525gcc aca gac gct tcc tgc tgc gca ggt gca ccc agc aca gag gca tca 1692 AlaThr Asp Ala Ser Cys Cys Ala Gly Ala Pro Ser Thr Glu Ala Ser 530 535 540ggc cag aca ggg ccc cag gag ccc acc act gcc act gcc tagagagctt 1741 GlyGln Thr Gly Pro Gln Glu Pro Thr Thr Ala Thr Ala 545 550 555 ggtgctggggtgtgccagga agggagcagg cagcccagcc aggcctggcc cagcccaggc 1801 tcccatgctaagcagtccgg tgctgaggcc aggatgttct gacctggctg gcacctgacc 1861 ctctgcagtcttggattttg tgggtcagtt ttacatgcat atggcacaca tgcaaggcct 1921 cacacatttgtgtctctaag tgtactgtgg gcttgcatcg ggggtgacga tggacagatg 1981 aagccagcggctcccttgtg agctgaagtc ttacggagga gacggcgtct gcactgccat 2041 cgcagtgacctgcaggacga gttccttgag ctttccctgc ctgctttgag gctgagaccc 2101 ctcccggcccttcagagctc ctgacaggtg atacacaccc agccttgacc gcacttctct 2161 tgggtagctgggctctccta gcctccccca gaggcgccat tgcttctctt gacttggaga 2221 ggggatgcccaggcgtggcc ttggcaggca ctgggagcta gtgattgggc tgctctcctg 2281 cctcgagcaggggcaggagt gtttctggtg ggatgatgcg ctcgctggtc aggagccccg 2341 tgggcgctgcttcccccgcc ctctggtgat gccaggacca ggccagtgat gcttctcagt 2401 agccttaccattcacaggtg cctctccagc ccgcacagtg agtgacaaga tcatccaaag 2461 gattccttctgaaggtgttc gtttcgtttt gttttgttgc acgtgacggt ttgtattgag 2521 gaccctctgaggaagagggg tgctgtagca gtggtccctg cgtgcctggc tccagtgtcc 2581 tgccctcccccccctcgcca tggctcctcg gccgccttgg tgctgaggtt tctgtttggt 2641 gagatcaggttgtctgttca gagagaagag gcgtctgatg gctttgccgc cagcttgcct 2701 gcgggcctcaatcccgggag gccgcccggt tcccgtcact gttgtccccg tgcagtgcgt 2761 tgctggtccccaggaccagc tgctcgtttg ctgtatgggt cagtttctgc ttcctgcccc 2821 ccactccacctaactgcaat ccttggggtt tccctggttc tcgtccctgg tacctctgtg 2881 cccaagaagtagccttcttt gggattcttg ttctgcccat gcgggagctg ctgctgtctg 2941 acaggtgaggcctgagactc agcggctgac agagctgcag agctctgcac ggtggctccc 3001 ggggcggcctctgtgtgctg cacaccgctg ctctgctggc actggccagt ctgtgcagag 3061 catttgagtactggctcagg agggagggct ctgctggcct cgagggacag cgccacgtct 3121 ccagctgggctcagggagag ccccagactg gctgcgtagg gtgcttgggg tttgcttctt 3181 gcagtatttcttggaagctg ttttgttgtc ctgctattcc ttcatcttcc acagtccacg 3241 ctcagcctttaacttggatc cctcacataa cagggttcat gagacccgca agtacgccca 3301 agctacgtatggctgaggcc agctggcagg tgaatggcac gccattgctg ctgctaatcc 3361 ctggcatatctttagttcac ctcgaaatgc ccccgccaca gtgcaagcag tgagtccacg 3421 tgccaccctgggctgaatcc caccccctgt gagtgttgcc cgagattgtg tctcttctga 3481 atgccttcactgggaatggc ctctgccgcc tcctgctcag ggaggctttc cccttccctc 3541 agcccctgtgccagactgag gtacaagaac cgccaagccc atgcaaggtg tggctaggcg 3601 ccagggtgcaggaaggaggc aggtagctgc ctgcaccctt gaaagccaag aggcctacgg 3661 tggcctccatcctggcttgc ctcacttcag ctacctcgca tagcccaggg gtggggctat 3721 tggattccagggtgggggga tgggaagctg cagggggcag gtggctctca ctaggcttcc 3781 cagctcaggaatgtgggcct caggtagggg agagcctttg ctccactcca cccatttgca 3841 ggcatctaggccagtctaga tggcgacccc ttctcttcct ctccattgac caaatcgtac 3901 ctgtctctccagctgctcgc ttgctctgct ttccaaagtc agcccaggta cccaggtgcc 3961 gcccacattggcctggaacc tggaccagag gcaagggagg tggcctatcc ttgagtgata 4021 gccagtgccttcctcacccg gtggcttcca tgcctgtgac ctcagattta ggaccaagag 4081 ctgtgttggtttcttacgtt gtgagctttc cctccagggg accacagcag gtgaggctcg 4141 gagcccagagcccttggcgc cgccagcagt aacttgtgtc cggaccttgt ccagctgagc 4201 gcttcgtgtatgactcagct tcgtgtgtga gtccagcgga gtgcgtcacg tgacctagac 4261 tcagcggtgtcagccgcact ttgatttgtt tgttttccat gaggtttttg gaccatgggc 4321 ttagctcaggcaacttttct gtaaggagaa tgttaacttt ctgtaaagat gcttatttaa 4381 ctaacgcctgcttcccccac tcccaaccag gtggccaccg agagctcacc aggaggccaa 4441 tagagctgctccagctctcc catcttgcac cgcacaaagg tggccgcccc agggacagcc 4501 aggcacctgcctgggggagg ggcttctctt ccttatggcc tggccatcta gattgtttaa 4561 agttgtgctgacagcttttt ttggtttttt ggtttttgtt tttgtttttg tttttgtttt 4621 tgtctacttttggtattcac aacagccagg gacttgattt tgatgtattt taagccacat 4681 taaataaagagtctgttgcc ttaaaaaaaa aaaaaaaaaa a 4722 15 1925 DNA Homo sapiens CDS(118)...(570) 15 gacgcctcag agcggaacag ggaagtgaat caggcgccgg gtagtgggttgctgggctgg 60 gcttgctgag gtagaggcag cgccaagaag aggcctttgc cgctggtcgggattggg atg 120 Met 1 tcg aag aac aca gtg tcg tcg gcc cgc ttc cgg aaggtg gac gtg gat 168 Ser Lys Asn Thr Val Ser Ser Ala Arg Phe Arg Lys ValAsp Val Asp 5 10 15 gaa tat gac gag aac aag ttc gtg gac gaa gaa gat gggggc gac ggc 216 Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp Gly GlyAsp Gly 20 25 30 cag gcc ggg ccc gac gag ggc gag gtg gac tcc tgc ctg cggcaa gga 264 Gln Ala Gly Pro Asp Glu Gly Glu Val Asp Ser Cys Leu Arg GlnGly 35 40 45 aac atg aca gct gcc cta cag gca gct ctg aag aac ccc cct atcaac 312 Asn Met Thr Ala Ala Leu Gln Ala Ala Leu Lys Asn Pro Pro Ile Asn50 55 60 65 acc aag agt cag gca gtg aag gac cgg gca ggc agc att gtc ttgaag 360 Thr Lys Ser Gln Ala Val Lys Asp Arg Ala Gly Ser Ile Val Leu Lys70 75 80 gtg ctc atc tct ttt aaa gct aat gat ata gaa aag gca gtt caa tct408 Val Leu Ile Ser Phe Lys Ala Asn Asp Ile Glu Lys Ala Val Gln Ser 8590 95 ctg gac aag aat ggt gtg gat ctc cta atg aag tat att tat aaa gga456 Leu Asp Lys Asn Gly Val Asp Leu Leu Met Lys Tyr Ile Tyr Lys Gly 100105 110 ttt gag agc ccg tct gac aat agc agt gct atg tta ctg caa tgg cat504 Phe Glu Ser Pro Ser Asp Asn Ser Ser Ala Met Leu Leu Gln Trp His 115120 125 gaa aag gca ctt gct gct gga gga gta ggg tcc att gtt cgt gtc ttg552 Glu Lys Ala Leu Ala Ala Gly Gly Val Gly Ser Ile Val Arg Val Leu 130135 140 145 act gca aga aaa act gtg tagtctggca ggaagtggat tatctgcctc 600Thr Ala Arg Lys Thr Val 150 gggagtggga attgctggta caaagaccaa aacaaccaaatgccaccgct gccctgtggg 660 tagcatctgt ttctctcagc tttgccttct tgctttttcatatctgtaaa gaaaaaaatt 720 acatatcagt tgtcccttta atgaaaattg ggataatatagaagaaattg tgttaaaata 780 gaagtgtttc atcctttcaa aaccatttca gtgatgtttataccaatctg tatatagtat 840 aatttacatt caagttttaa ttgtgcaact tttaaccctgttggctggtt tttggttctg 900 tttggttttg tattattttt aactaatact gaaaaatttggtcagaattt gaggccagtt 960 tcctagctca ttgctagtca ggaaatgata tttataaaaaatatgagaga ctggcagcta 1020 ttaacattgc aaaactggac catatttccc ttatttaataagcaaaatat gtttttggaa 1080 taagtggtgg gtgaatacca ctgctaagtt atagctttgtttttgcttgc ctcctcatta 1140 tctgtactgt gggtttaagt atgctacttt ctctcagcatccaataatca tggcccctca 1200 atttatttgt ggtcacgcag ggttcagagc aagaagtcttgctttataca aatgtatcca 1260 taaaatatca gagcttgttg ggcatgaaca tcaaacttttgttccactaa tatggctctg 1320 tttggaaaaa actgcaaatc agaaagaatg atttgcagaaagaaagaaaa actatggtgt 1380 aatttaaact ctgggcagcc tctgaatgaa atgctactttctttagaaat ataatagctg 1440 ccttagacat tatgaggtat acaactagta tttaagataccatttaatat gccccgtaaa 1500 tgtcttcagt gttcttcagg gtagttggga tctcaaaagatttggttcag atccaaacaa 1560 atacacattc tgtgttttag ctcagtgttt tctaaaaaaagaaactgcca cacagcaaaa 1620 aattgtttac tttgttggac aaaccaaatc agttctcaaaaaatgaccgg tgcttataaa 1680 aagttataaa tatcgagtag ctctaaaaca aaccacctgaccaagaggga agtgagcttg 1740 tgcttagtat ttacattgga tgccagtttt gtaatcactgacttatgtgc aaactggtgc 1800 agaaattcta taaactcttt gctgtttttg atacctgctttttgtttcat tttgttttgt 1860 tttgtaaaaa tgataaaact tcagaaaata aaatgtcagtgttgaataat taaaaaaaaa 1920 aaaaa 1925 16 1208 DNA Homo sapiens CDS(1)...(651) 16 gaa gag cga gta ctt gag aaa gaa gag gaa gaa gat gat gatgaa gat 48 Glu Glu Arg Val Leu Glu Lys Glu Glu Glu Glu Asp Asp Asp GluAsp 1 5 10 15 gaa gat gaa gaa gat gat gtg tca gag ggc tct gaa gtg cccgag agt 96 Glu Asp Glu Glu Asp Asp Val Ser Glu Gly Ser Glu Val Pro GluSer 20 25 30 gac cgt cct gca ggt gcc cag cac cac cag ctt aac ggc gag cgggga 144 Asp Arg Pro Ala Gly Ala Gln His His Gln Leu Asn Gly Glu Arg Gly35 40 45 cct cag agt gcc aag gag agg gtc aag gag tgg acc ccc tgc gga ccg192 Pro Gln Ser Ala Lys Glu Arg Val Lys Glu Trp Thr Pro Cys Gly Pro 5055 60 cac cag ggc cag gat gaa ggg cgg ggg cca gcc ccg ggc agc ggc acc240 His Gln Gly Gln Asp Glu Gly Arg Gly Pro Ala Pro Gly Ser Gly Thr 6570 75 80 cgc cag gtg ttc tcc atg gca gcc atg aac aag gaa ggg gga aca gct288 Arg Gln Val Phe Ser Met Ala Ala Met Asn Lys Glu Gly Gly Thr Ala 8590 95 tct gtt gcc acc ggg cca gac tcc ccg tcc ccc gtg cct ttg ccc cca336 Ser Val Ala Thr Gly Pro Asp Ser Pro Ser Pro Val Pro Leu Pro Pro 100105 110 ggc aaa cca gcc cta cct ggg gcc gac ggg acc ccc ttt ggc tgt cct384 Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro 115120 125 ccc ggg cgc aaa gag aag cca tct gat ccc gtc gag tgg acc gtg atg432 Pro Gly Arg Lys Glu Lys Pro Ser Asp Pro Val Glu Trp Thr Val Met 130135 140 gat gtc gtc gaa tat ttt act gag gct gga ttc ccg gag cag gcg aca480 Asp Val Val Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr 145150 155 160 gct ttc caa gag cag gaa att gat ggc aaa tct ttg ctg ctc atgcag 528 Ala Phe Gln Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln165 170 175 cgc aca gat gtg ctc acc ggc ctg tcc atc cgc ctc ggg cca gccctg 576 Arg Thr Asp Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala Leu180 185 190 aaa atc tac gag cac cac atc aag gtg ctt cag caa ggc cac tttgag 624 Lys Ile Tyr Glu His His Ile Lys Val Leu Gln Gln Gly His Phe Glu195 200 205 gat gat gac ccc gat ggc ttc tta ggc tgagcgccca gcctcacccc671 Asp Asp Asp Pro Asp Gly Phe Leu Gly 210 215 tgccccagcc cattccggcccccatctcac ccaagatccc ccagagtcca ggagctggac 731 ggggacaccc tcagccctcataacagattc caaggagagg gcaccctctt gtccttatct 791 ttgccccttg tgtctgtctcacacacatct gctcctcagc acgtcggtgt ggggagggga 851 ttgctcctta aaccccaggtggctgaccct ccccacccag tccaggacat tttaggaaaa 911 aaaaaatgaa atgtggggggcttctcatct ccccaagatc ctcttccgtt cagccagatg 971 tttcctgtat aaatgtttggatctgcctgt ttattttggt gggtggtctt tcctccctcc 1031 cctaccaccc atgccccccttctcagtctg cccctggcct ccagccccta ggggactagc 1091 tgggttgggg ttcctcgggccttttctctc ctccctcttt tctttctgtt gattgtcgct 1151 ccagctggct gtattgctttttaatattgc accgaaggtt ttttaaataa aatttta 1208 17 4697 DNA Homo sapiensCDS (3)...(1592) 17 ca aaa agc agc cca gga caa ccg gaa gca gga ccc gaggga gcc cag 47 Lys Ser Ser Pro Gly Gln Pro Glu Ala Gly Pro Glu Gly AlaGln 1 5 10 15 gag cgg ccc agc cag gcg gct cct gca gta gaa gca gaa ggtccc ggc 95 Glu Arg Pro Ser Gln Ala Ala Pro Ala Val Glu Ala Glu Gly ProGly 20 25 30 agc agc cag gct cct cgg aag ccg gag ggg gct caa gcc aga acggct 143 Ser Ser Gln Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala Arg Thr Ala35 40 45 cag tct ggg gcc ctt cgt gat gtc tct gag gag ctg agc cgc caa ctg191 Gln Ser Gly Ala Leu Arg Asp Val Ser Glu Glu Leu Ser Arg Gln Leu 5055 60 gaa gac ata ctg agc aca tac tgt gtg gac aat aac cag ggg ggc ccc239 Glu Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly Gly Pro 6570 75 ggc gag gat ggg gca cag ggt gag ccg gct gaa ccc gaa gat gca gag287 Gly Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu Asp Ala Glu 8085 90 95 aag tcc cgg acc tat gtg gca agg aat ggg gag cct gaa cca act cca335 Lys Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu Pro Glu Pro Thr Pro 100105 110 gta gtc tat gga gag aag gaa ccc tcc aag ggg gat cca aac aca gaa383 Val Val Tyr Gly Glu Lys Glu Pro Ser Lys Gly Asp Pro Asn Thr Glu 115120 125 gag atc cgg cag agt gac gag gtc gga gac cga gac cat cga agg cca431 Glu Ile Arg Gln Ser Asp Glu Val Gly Asp Arg Asp His Arg Arg Pro 130135 140 cag gag aag aaa aaa gcc aag ggt ttg ggg aag gag atc acg ttg ctg479 Gln Glu Lys Lys Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu 145150 155 atg cag aca ttg aat act ctg agt acc cca gag gag aag ctg gct gct527 Met Gln Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala 160165 170 175 ctg tgc aag aag tat gct gaa ctg ctg gag gag cac cgg aat tcacag 575 Leu Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn Ser Gln180 185 190 aag cag atg aag ctc cta cag aaa aag cag agc cag ctg gtg caagag 623 Lys Gln Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val Gln Glu195 200 205 aag gac cac ctg cgc ggt gag cac agc aag gcc gtc ctg gcc cgcagc 671 Lys Asp His Leu Arg Gly Glu His Ser Lys Ala Val Leu Ala Arg Ser210 215 220 aag ctt gag agc cta tgc cgt gag ctg cag cgg cac aac cgc tccctc 719 Lys Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn Arg Ser Leu225 230 235 aag gaa gaa ggt gtg cag cgg gcc cgg gag gag gag gag aag cgcaag 767 Lys Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu Glu Lys Arg Lys240 245 250 255 gag gtg acc tcg cac ttc cag gtg aca ctg aat gac att cagctg cag 815 Glu Val Thr Ser His Phe Gln Val Thr Leu Asn Asp Ile Gln LeuGln 260 265 270 atg gaa cag cac aat gag cgc aac tcc aag ctg cgc caa gagaac atg 863 Met Glu Gln His Asn Glu Arg Asn Ser Lys Leu Arg Gln Glu AsnMet 275 280 285 gag ctg gct gag agg ctc aag aag ctg att gag cag tat gagctg cgc 911 Glu Leu Ala Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr Glu LeuArg 290 295 300 gag gag cat atc gac aaa gtc ttc aaa cac aag gac cta caacag cag 959 Glu Glu His Ile Asp Lys Val Phe Lys His Lys Asp Leu Gln GlnGln 305 310 315 ctg gtg gat gcc aag ctc cag cag gcc cag gag atg cta aaggag gca 1007 Leu Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met Leu Lys GluAla 320 325 330 335 gaa gag cgg cac cag cgg gag aag gat ttt ctc ctg aaagag gca gta 1055 Glu Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu Lys GluAla Val 340 345 350 gag tcc cag agg atg tgt gag ctg atg aag cag caa gagacc cac ctg 1103 Glu Ser Gln Arg Met Cys Glu Leu Met Lys Gln Gln Glu ThrHis Leu 355 360 365 aag caa cag ctt gcc cta tac aca gag aag ttt gag gagttc cag aac 1151 Lys Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe Glu Glu PheGln Asn 370 375 380 aca ctt tcc aaa agc agc gag gta ttc acc aca ttc aagcag gag atg 1199 Thr Leu Ser Lys Ser Ser Glu Val Phe Thr Thr Phe Lys GlnGlu Met 385 390 395 gaa aag atg act aag aag atc aag aag ctg gag aaa gaaacc acc atg 1247 Glu Lys Met Thr Lys Lys Ile Lys Lys Leu Glu Lys Glu ThrThr Met 400 405 410 415 tac cgg tcc cgg tgg gag agc agc aac aag gcc ctgctt gag atg gct 1295 Tyr Arg Ser Arg Trp Glu Ser Ser Asn Lys Ala Leu LeuGlu Met Ala 420 425 430 gag gag aaa aca gtc cgg gat aaa gaa ctg gag ggcctg cag gta aaa 1343 Glu Glu Lys Thr Val Arg Asp Lys Glu Leu Glu Gly LeuGln Val Lys 435 440 445 atc caa cgg ctg gag aag ctg tgc cgg gca ctg cagaca gag cgc aat 1391 Ile Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu Gln ThrGlu Arg Asn 450 455 460 gac ctg aac aag agg gta cag gac ctg agt gct ggtggc cag ggc tcc 1439 Asp Leu Asn Lys Arg Val Gln Asp Leu Ser Ala Gly GlyGln Gly Ser 465 470 475 ctc act gac agt ggc cct gag agg agg cca gag gggcct ggg gct caa 1487 Leu Thr Asp Ser Gly Pro Glu Arg Arg Pro Glu Gly ProGly Ala Gln 480 485 490 495 gca ccc agc tcc ccc agg gtc aca gaa gcg ccttgc tac cca gga gca 1535 Ala Pro Ser Ser Pro Arg Val Thr Glu Ala Pro CysTyr Pro Gly Ala 500 505 510 ccg agc aca gaa gca tca ggc cag act ggg cctcaa gag ccc acc tcc 1583 Pro Ser Thr Glu Ala Ser Gly Gln Thr Gly Pro GlnGlu Pro Thr Ser 515 520 525 gcc agg gcc tagagagcct ggtgttgggt catgctgggaagggagcggc 1632 Ala Arg Ala 530 agcccagcca ggcctggccc ataaaaggctcccatgctga gcagcccatt gctgaagcca 1692 ggatgttctt gacctggctg gcatctggcacttgcaattt tggattttgt gggtcagttt 1752 tacgtacata gggcattttg caaggccttgcaaatgcatt tatacctgta agtgtacagt 1812 gggcttgcat tggggatggg ggtgtgtacagatgaagtca gtggcttgtc tgtgagctga 1872 agagtcttga gaggggctgt catctgtagctgccatcaca gtgagttggc agaagtgact 1932 tgagcatttc tctgtctgat ttgaggctcagacccctccc tgccctttca gagctcaaaa 1992 caagtaatac accaaggtct tgactgcatttgtcttgtga gcagggcttg cttggtcagc 2052 tcaggccctc ctagctgctt ggaggctcctttgattctct agacctggaa aaggtgtccc 2112 taggcagagc cctggcaggg cgctcagagctgggatttcc tgcctggaac aagggacctg 2172 gagaatgttt ttgcgtggga tgatgtgctggtcaggagcc ccttgggcat cgcttcccct 2232 gccctttggt agtgccagga ccaggccaatgatgcttctc agtagcctta tcattcacag 2292 gtgcctctct agcctgcaca aatgattgacaagagatcac ccaaaggatt atttctgaag 2352 gtgttttttt ctttatttct ttttctttttttttttttct ttttcttttt tttttgcaca 2412 tgacagtgtt tgtattgagg accttccaaggaaaagggat gctgtaccag tggtgcctgg 2472 gtgcctggcc tccagtgtcc cacctccttcaccaccccac ttggctcctt tgccatcttg 2532 atgctgaggt ttcctgtttg gtgagatcaggttgtttgtg gtaaaagaaa ggaaagggct 2592 tctgatggct ttgccacaag cttacctgtgggtttcagtc ctgagaggcc accaccagtt 2652 cccatcagca ctgtctccat gcagcagttgctgggtccca tgtccagctg cctctttggc 2712 ttcatgggtt tttctgcttc ctgcccccacccccacatgt gcaatcctca agatttgtcc 2772 tgattctatt tcctggcacc tccctgcctgtccttgggga ttctacttct tcctgtgtgg 2832 ggcccatagc tgttgtctaa caggtaagaaatgaaattga actattgact gggccccaga 2892 aatccataaa atggctgcag acagttgtttctgtgtcctg ttctaccccc actccagtac 2952 ataactacta tgtactgtgt agagccattctatatgctga atgttctgct gttgcaaact 3012 tgccagggta ttagccagtg tttgtgccaagcagttttcg gggacaacag aatgactcag 3072 accaagatgg ataggatggt tagggctttgcttcttgctg tttttctttg aactagtcat 3132 tgtcctgcag gtcccttcat cttccatacctagcccactc ttttagccct taccttaaat 3192 ctctcagata agttggttca caaagaatgttaagtactga atcatgtgtg actgagacca 3252 gagatggcaa atgaatggca caccatttctccttctcctg ccccagggca ggtaccactg 3312 atctgcatca gagttgcctg ctattctctggtgtatcctt cacatctagg tgccctcaag 3372 cagctgtgtg agtgttgaga tctctgccatctctggctga gatactgctg tcctgtgaag 3432 tgtttcccat gacctttttc ttcccctttgaatccctctt gtctggagta gtccttgcct 3492 tcttcttgct ccagtaggcc ttttccttaccccagccctt gtgccaggct aagctggtac 3552 aagagctgcc aactcacaga gttttgctaggcgagagagg tgcagggaag aggcagaggt 3612 atgcaccttc ccccttgaag agaggggaaaggcctacagt ggcccacata attgcctgac 3672 tcacacttca gctacctctt aatgcctgtggagggactgg agctgctgga tcccagtgtg 3732 gtggtgtagg aggccacagt gagcaggtggccccagctgg gtttcccagg tcaggaatgt 3792 gggccccagg caaggtgcag cctttgctcacagctccatc catgtctaga ccttcaggcc 3852 agtctgcaga tgaggttccc tacctttttcttctcttcat tgaccaaatc aaccaatcac 3912 tacagctgct ctgcttctgc tttccaaagtagcccaggtc ctgggccaga tgcaggggag 3972 gtgcctatcc atgagtgaag gccagtgtcttcctcacctg ggtggtccca cacttgtgac 4032 cctcagtttt aggacccaag atctgtgttggtttcttaga ttgctagctt ttcctccagg 4092 ggaccacagc aggtgaagct caagagcgcatggctctgct aatagtaaat tgttttcagg 4152 gccttgtcca gctgagagct tcatgtccaccagattctga gaggtgtcag cagcactttt 4212 tttttttatt tgttgtttgt tttccatgaggttatcggac catgggctga gctcaggcac 4272 tttctgtagg agactgttat ttctgtaaagatggttattt aaccctcctc caccccatca 4332 cggtggccct gagggctgac ccggaggccagtggagctgc ctggtgtcca cgggggaggg 4392 ccaaggcctg ctgagctgat tctccagctgctgccccagc ctttccgcct tgcacagcac 4452 agaggtggtc accccaggga cagccaggcacctgctcctc ttgcccttcc tgggggaaag 4512 gagctgcctt ctgtccctgt aactgctttccttatggccc aacccggcca ctcagacttg 4572 tttgaagctg cactggcagc ttttttgtctcctttgggta ttcacaacag ccagggactt 4632 gattttgatg tattttaaac cacattaaataaagagtctg ttgccttaaa aaaaaaaaaa 4692 aaaaa 4697 18 60 DNA Homo sapiensCDS (1)...(60) 18 gtg gac gtg gat gag tac gac gag aac aag ttc gtg gacgag gaa gac 48 Val Asp Val Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp GluGlu Asp 1 5 10 15 ggc ggc gac ggc 60 Gly Gly Asp Gly 20 19 15 PRT Homosapiens 19 Glu Glu Glu Glu Asp Asp Asp Glu Asp Glu Asp Glu Glu Asp Asp 15 10 15 20 26 PRT Homo sapiens 20 Glu Glu Glu Glu Asp Asp Asp Glu AspGlu Asp Glu Glu Asp Asp Val 1 5 10 15 Ser Glu Gly Ser Glu Val Pro GluSer Asp 20 25 21 11 PRT Homo sapiens 21 Val Ser Glu Gly Ser Glu Val ProGlu Ser Asp 1 5 10 22 10 PRT Homo sapiens 22 Glu Asp Asp Asp Pro Asp GlyPhe Leu Gly 1 5 10 23 30 PRT Oryctolagus cuniculus 23 Val Asp Val AspGlu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp 1 5 10 15 Gly Gly AspGly Gln Ala Gly Pro Asp Glu Gly Glu Val Asp 20 25 30 24 6 PRTOryctolagus cuniculus 24 Asp Glu Gly Glu Val Asp 1 5 25 16 PRTOryctolagus cuniculus 25 Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu AspAsp Asp Asp Asp 1 5 10 15 26 28 PRT Oryctolagus cuniculus 26 Glu Glu GluGlu Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Asp Asp 1 5 10 15 Val ValSer Glu Gly Ser Glu Val Pro Glu Ser Asp 20 25 27 12 PRT Oryctolaguscuniculus 27 Val Val Ser Glu Gly Ser Glu Val Pro Glu Ser Asp 1 5 10 2810 PRT Oryctolagus cuniculus 28 Pro Pro Gly Lys Pro Ala Leu Pro Gly Ala1 5 10 29 15 PRT Oryctolagus cuniculus 29 Glu Asp Gly Val Gln Gly GluPro Pro Glu Pro Glu Asp Ala Glu 1 5 10 15 30 45 DNA Homo sapiens 30gaagaggaag aagatgatga tgaagatgaa gatgaagaag atgat 45 31 78 DNA Homosapiens 31 gaagaggaag aagatgatga tgaagatgaa gatgaagaag atgatgtgtcagagggctct 60 gaagtgcccg agagtgac 78 32 33 DNA Homo sapiens 32gtgtcagagg gctctgaagt gcccgagagt gac 33 33 30 DNA Homo sapiens 33gaggatgatg accccgatgg cttcttaggc 30 34 90 DNA Homo sapiens 34 gtggacgtggatgaatatga cgagaacaag ttcgtggacg aagaagatgg gggcgacggc 60 caggccgggcccgacgaggg cgaggtggac 90 35 18 DNA Homo sapiens 35 gacgagggcg aggtggac18 36 48 DNA Homo sapiens 36 gaggaggagg aggaggagga ggaagacgac gaggacgacgacgacgac 48 37 84 DNA Homo sapiens 37 gaggaggagg aggaggagga ggaagacgacgaggacgacg acgacgacgt cgtgtccgag 60 ggctcggagg tgcccgagag cgat 84 38 36DNA Homo sapiens 38 gtcgtgtccg agggctcgga ggtgcccgag agcgat 36 39 30 DNAHomo sapiens 39 ccccccggga agccagccct cccaggagcc 30 40 45 DNA Homosapiens 40 gaggatgggg tccagggtga gccccctgaa cctgaagatg cagag 45 41 7 PRTHomo sapiens 41 Arg Asp Val Ser Glu Glu Leu 1 5 42 21 DNA Homo sapiens42 cgtgatgtct ctgaggagct g 21 43 538 PRT Homo sapiens 43 Met Ala Gly ProPro Ala Leu Pro Pro Pro Glu Thr Ala Ala Ala Ala 1 5 10 15 Thr Thr AlaAla Ala Ala Ser Ser Ser Ala Ala Ser Pro His Tyr Gln 20 25 30 Glu Trp IleLeu Asp Thr Ile Asp Ser Leu Arg Ser Arg Lys Ala Arg 35 40 45 Pro Asp LeuGlu Arg Ile Cys Arg Met Val Arg Arg Arg His Gly Pro 50 55 60 Glu Pro GluArg Thr Arg Ala Glu Leu Glu Lys Leu Ile Gln Gln Arg 65 70 75 80 Ala ValLeu Arg Val Ser Tyr Lys Gly Ser Ile Ser Tyr Arg Asn Ala 85 90 95 Ala ArgVal Gln Pro Pro Arg Arg Gly Ala Thr Pro Pro Ala Pro Pro 100 105 110 ArgAla Pro Arg Gly Ala Pro Ala Ala Ala Ala Ala Ala Ala Pro Pro 115 120 125Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala Pro Val Ala Ala Ala Ala 130 135140 Pro Ala Arg Ala Pro Arg Ala Ala Ala Ala Ala Ala Thr Ala Pro Pro 145150 155 160 Ser Pro Gly Pro Ala Gln Pro Gly Pro Arg Ala Gln Arg Ala AlaPro 165 170 175 Leu Ala Ala Pro Pro Pro Ala Pro Ala Ala Pro Pro Ala ValAla Pro 180 185 190 Pro Ala Gly Pro Arg Arg Ala Pro Pro Pro Ala Val AlaAla Arg Glu 195 200 205 Pro Pro Leu Pro Pro Pro Pro Gln Pro Pro Ala ProPro Gln Gln Gln 210 215 220 Gln Pro Pro Pro Pro Gln Pro Gln Pro Pro ProGlu Gly Gly Ala Val 225 230 235 240 Arg Ala Gly Gly Ala Ala Arg Pro ValSer Leu Arg Glu Val Val Arg 245 250 255 Tyr Leu Gly Gly Ser Gly Gly AlaGly Gly Arg Leu Thr Arg Gly Arg 260 265 270 Val Gln Gly Leu Leu Glu GluGlu Ala Ala Ala Arg Gly Arg Leu Glu 275 280 285 Arg Thr Arg Leu Gly AlaLeu Ala Leu Pro Arg Gly Asp Arg Pro Gly 290 295 300 Arg Ala Pro Pro AlaAla Ser Ala Arg Pro Ser Arg Ser Lys Arg Gly 305 310 315 320 Gly Glu GluArg Val Leu Glu Lys Glu Glu Glu Glu Asp Asp Asp Glu 325 330 335 Asp GluAsp Glu Glu Asp Asp Val Ser Glu Gly Ser Glu Val Pro Glu 340 345 350 SerAsp Arg Pro Ala Gly Ala Gln His His Gln Leu Asn Gly Glu Arg 355 360 365Gly Pro Gln Ser Ala Lys Glu Arg Val Lys Glu Trp Thr Pro Cys Gly 370 375380 Pro His Gln Gly Gln Asp Glu Gly Arg Gly Pro Ala Pro Gly Ser Gly 385390 395 400 Thr Arg Gln Val Phe Ser Met Ala Ala Met Asn Lys Glu Gly GlyThr 405 410 415 Ala Ser Val Ala Thr Gly Pro Asp Ser Pro Ser Pro Val ProLeu Pro 420 425 430 Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp Gly Thr ProPhe Gly Cys 435 440 445 Pro Pro Gly Arg Lys Glu Lys Pro Ser Asp Pro ValGlu Trp Thr Val 450 455 460 Met Asp Val Val Glu Tyr Phe Thr Glu Ala GlyPhe Pro Glu Gln Ala 465 470 475 480 Thr Ala Phe Gln Glu Gln Glu Ile AspGly Lys Ser Leu Leu Leu Met 485 490 495 Gln Arg Thr Asp Val Leu Thr GlyLeu Ser Ile Arg Leu Gly Pro Ala 500 505 510 Leu Lys Ile Tyr Glu His HisIle Lys Val Leu Gln Gln Gly His Phe 515 520 525 Glu Asp Asp Asp Pro AspGly Phe Leu Gly 530 535 44 546 PRT Homo sapiens 44 Met Lys Asn Gln AspLys Lys Asn Gly Ala Ala Lys Gln Ser Asn Pro 1 5 10 15 Lys Ser Ser ProGly Gln Pro Glu Ala Gly Pro Glu Gly Ala Gln Glu 20 25 30 Arg Pro Ser GlnAla Ala Pro Ala Val Glu Ala Glu Gly Pro Gly Ser 35 40 45 Ser Gln Ala ProArg Lys Pro Glu Gly Ala Gln Ala Arg Thr Ala Gln 50 55 60 Ser Gly Ala LeuArg Asp Val Ser Glu Glu Leu Ser Arg Gln Leu Glu 65 70 75 80 Asp Ile LeuSer Thr Tyr Cys Val Asp Asn Asn Gln Gly Gly Pro Gly 85 90 95 Glu Asp GlyAla Gln Gly Glu Pro Ala Glu Pro Glu Asp Ala Glu Lys 100 105 110 Ser ArgThr Tyr Val Ala Arg Asn Gly Glu Pro Glu Pro Thr Pro Val 115 120 125 ValAsn Gly Glu Lys Glu Pro Ser Lys Gly Asp Pro Asn Thr Glu Glu 130 135 140Ile Arg Gln Ser Asp Glu Val Gly Asp Arg Asp His Arg Arg Pro Gln 145 150155 160 Glu Lys Lys Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr Leu Leu Met165 170 175 Gln Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala AlaLeu 180 185 190 Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn SerGln Lys 195 200 205 Gln Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu ValGln Glu Lys 210 215 220 Asp His Leu Arg Gly Glu His Ser Lys Ala Val LeuAla Arg Ser Lys 225 230 235 240 Leu Glu Ser Leu Cys Arg Glu Leu Gln ArgHis Asn Arg Ser Leu Lys 245 250 255 Glu Glu Gly Val Gln Arg Ala Arg GluGlu Glu Glu Lys Arg Lys Glu 260 265 270 Val Thr Ser His Phe Gln Val ThrLeu Asn Asp Ile Gln Leu Gln Met 275 280 285 Glu Gln His Asn Glu Arg AsnSer Lys Leu Arg Gln Glu Asn Met Glu 290 295 300 Leu Ala Glu Arg Leu LysLys Leu Ile Glu Gln Tyr Glu Leu Arg Glu 305 310 315 320 Glu His Ile AspLys Val Phe Lys His Lys Asp Leu Gln Gln Gln Leu 325 330 335 Val Asp AlaLys Leu Gln Gln Ala Gln Glu Met Leu Lys Glu Ala Glu 340 345 350 Glu ArgHis Gln Arg Glu Lys Asp Phe Leu Leu Lys Glu Ala Val Glu 355 360 365 SerGln Arg Met Cys Glu Leu Met Lys Gln Gln Glu Thr His Leu Lys 370 375 380Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe Glu Glu Phe Gln Asn Thr 385 390395 400 Leu Ser Lys Ser Ser Glu Val Phe Thr Thr Phe Lys Gln Glu Met Glu405 410 415 Lys Met Thr Lys Lys Ile Lys Lys Leu Glu Lys Glu Thr Thr MetTyr 420 425 430 Arg Ser Arg Trp Glu Ser Ser Asn Lys Ala Leu Leu Glu MetAla Glu 435 440 445 Glu Lys Thr Val Arg Asp Lys Glu Leu Glu Gly Leu GlnVal Lys Ile 450 455 460 Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu Gln ThrGlu Arg Asn Asp 465 470 475 480 Leu Asn Lys Arg Val Gln Asp Leu Ser AlaGly Gly Gln Gly Ser Leu 485 490 495 Thr Asp Ser Gly Pro Glu Arg Arg ProGlu Gly Pro Gly Ala Gln Ala 500 505 510 Pro Ser Ser Pro Arg Val Thr GluAla Pro Cys Tyr Pro Gly Ala Pro 515 520 525 Ser Thr Glu Ala Ser Gly GlnThr Gly Pro Gln Glu Pro Thr Ser Ala 530 535 540 Arg Ala 545 45 1614 DNAHomo sapiens CDS (1)...(1614) 45 atg gcg ggg ccc ccg gcc cta ccc ccg ccggag acg gcg gcg gcc gcc 48 Met Ala Gly Pro Pro Ala Leu Pro Pro Pro GluThr Ala Ala Ala Ala 1 5 10 15 acc acg gcg gcc gcc gcc tcg tcg tcc gccgct tcc ccg cac tac caa 96 Thr Thr Ala Ala Ala Ala Ser Ser Ser Ala AlaSer Pro His Tyr Gln 20 25 30 gag tgg atc ctg gac acc atc gac tcg ctg cgctcg cgc aag gcg cgg 144 Glu Trp Ile Leu Asp Thr Ile Asp Ser Leu Arg SerArg Lys Ala Arg 35 40 45 ccg gac ctg gag cgc atc tgc cgg atg gtg cgg cggcgg cac ggc ccg 192 Pro Asp Leu Glu Arg Ile Cys Arg Met Val Arg Arg ArgHis Gly Pro 50 55 60 gag ccg gag cgc acg cgc gcc gag ctc gag aaa ctg atccag cag cgc 240 Glu Pro Glu Arg Thr Arg Ala Glu Leu Glu Lys Leu Ile GlnGln Arg 65 70 75 80 gcc gtg ctc cgg gtc agc tac aag ggg agc atc tcg taccgc aac gcg 288 Ala Val Leu Arg Val Ser Tyr Lys Gly Ser Ile Ser Tyr ArgAsn Ala 85 90 95 gcg cgc gtc cag ccg ccc cgg cgc gga gcc acc ccg ccg gccccg ccg 336 Ala Arg Val Gln Pro Pro Arg Arg Gly Ala Thr Pro Pro Ala ProPro 100 105 110 cgc gcc ccc cgc ggg gcc ccc gcc gcc gcc gcc gcc gcc gcgccg ccg 384 Arg Ala Pro Arg Gly Ala Pro Ala Ala Ala Ala Ala Ala Ala ProPro 115 120 125 ccc acg ccc gcc ccg ccg cca ccg ccc gcg ccc gtc gcc gccgcc gcc 432 Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala Pro Val Ala Ala AlaAla 130 135 140 ccg gcc cgg gcg ccc cgc gcg gcc gcc gcc gcc gcc aca gcgccc ccc 480 Pro Ala Arg Ala Pro Arg Ala Ala Ala Ala Ala Ala Thr Ala ProPro 145 150 155 160 tcg cct ggc ccc gcg cag ccg ggc ccc cgc gcg cag cgggcc gcg ccc 528 Ser Pro Gly Pro Ala Gln Pro Gly Pro Arg Ala Gln Arg AlaAla Pro 165 170 175 ctg gcc gcg ccg ccg ccc gcg cca gcc gct ccc ccg gcggtg gcg ccc 576 Leu Ala Ala Pro Pro Pro Ala Pro Ala Ala Pro Pro Ala ValAla Pro 180 185 190 ccg gcc ggc ccg cgc cgc gcc ccc ccg ccc gcc gtc gccgcc cgg gag 624 Pro Ala Gly Pro Arg Arg Ala Pro Pro Pro Ala Val Ala AlaArg Glu 195 200 205 ccg ccg ctg ccg ccg ccg cca cag ccg ccg gcg ccg ccacag cag cag 672 Pro Pro Leu Pro Pro Pro Pro Gln Pro Pro Ala Pro Pro GlnGln Gln 210 215 220 cag ccg ccg ccg ccg cag cca cag ccg ccg ccg gag gggggc gcg gtg 720 Gln Pro Pro Pro Pro Gln Pro Gln Pro Pro Pro Glu Gly GlyAla Val 225 230 235 240 cgg gcc ggc ggc gcg gcg cgg ccc gtg agc ctg cgggaa gtc gtg cgc 768 Arg Ala Gly Gly Ala Ala Arg Pro Val Ser Leu Arg GluVal Val Arg 245 250 255 tac ctc ggg ggc agc ggc ggc gcc ggc ggt cgc ctaacc cgc ggc cgc 816 Tyr Leu Gly Gly Ser Gly Gly Ala Gly Gly Arg Leu ThrArg Gly Arg 260 265 270 gtg cag ggg ctg ctg gag gag gag gcg gcg gct cgaggc cgt ctg gag 864 Val Gln Gly Leu Leu Glu Glu Glu Ala Ala Ala Arg GlyArg Leu Glu 275 280 285 cgc acc cgt ctc gga gcg ctt gcg ctg ccc cgc ggggac agg ccc gga 912 Arg Thr Arg Leu Gly Ala Leu Ala Leu Pro Arg Gly AspArg Pro Gly 290 295 300 cgg gcg ccg ccg gcc gcc agc gcc cgc ccg tct cgcagc aag aga ggt 960 Arg Ala Pro Pro Ala Ala Ser Ala Arg Pro Ser Arg SerLys Arg Gly 305 310 315 320 gga gaa gag cga gta ctt gag aaa gaa gag gaagaa gat gat gat gaa 1008 Gly Glu Glu Arg Val Leu Glu Lys Glu Glu Glu GluAsp Asp Asp Glu 325 330 335 gat gaa gat gaa gaa gat gat gtg tca gag ggctct gaa gtg ccc gag 1056 Asp Glu Asp Glu Glu Asp Asp Val Ser Glu Gly SerGlu Val Pro Glu 340 345 350 agt gac cgt cct gca ggt gcc cag cac cac cagctt aac ggc gag cgg 1104 Ser Asp Arg Pro Ala Gly Ala Gln His His Gln LeuAsn Gly Glu Arg 355 360 365 gga cct cag agt gcc aag gag agg gtc aag gagtgg acc ccc tgc gga 1152 Gly Pro Gln Ser Ala Lys Glu Arg Val Lys Glu TrpThr Pro Cys Gly 370 375 380 ccg cac cag ggc cag gat gaa ggg cgg ggg ccagcc ccg ggc agc ggc 1200 Pro His Gln Gly Gln Asp Glu Gly Arg Gly Pro AlaPro Gly Ser Gly 385 390 395 400 acc cgc cag gtg ttc tcc atg gca gcc atgaac aag gaa ggg gga aca 1248 Thr Arg Gln Val Phe Ser Met Ala Ala Met AsnLys Glu Gly Gly Thr 405 410 415 gct tct gtt gcc acc ggg cca gac tcc ccgtcc ccc gtg cct ttg ccc 1296 Ala Ser Val Ala Thr Gly Pro Asp Ser Pro SerPro Val Pro Leu Pro 420 425 430 cca ggc aaa cca gcc cta cct ggg gcc gacggg acc ccc ttt ggc tgt 1344 Pro Gly Lys Pro Ala Leu Pro Gly Ala Asp GlyThr Pro Phe Gly Cys 435 440 445 ccg ccc ggg cgc aaa gag aag cca tct gatccc gtc gag tgg acc gtg 1392 Pro Pro Gly Arg Lys Glu Lys Pro Ser Asp ProVal Glu Trp Thr Val 450 455 460 atg gat gtc gtc gaa tat ttt act gag gctgga ttc ccg gag cag gcg 1440 Met Asp Val Val Glu Tyr Phe Thr Glu Ala GlyPhe Pro Glu Gln Ala 465 470 475 480 aca gct ttc caa gag cag gaa att gatggc aaa tct ttg ctg ctc atg 1488 Thr Ala Phe Gln Glu Gln Glu Ile Asp GlyLys Ser Leu Leu Leu Met 485 490 495 cag cgc aca gat gtg ctc acc ggc ctgtcc atc cgc ctc ggg cca gcc 1536 Gln Arg Thr Asp Val Leu Thr Gly Leu SerIle Arg Leu Gly Pro Ala 500 505 510 ctg aaa atc tac gag cac cac atc aaggtg ctt cag caa ggc cac ttt 1584 Leu Lys Ile Tyr Glu His His Ile Lys ValLeu Gln Gln Gly His Phe 515 520 525 gag gat gat gac ccc gat ggc ttc ttaggc 1614 Glu Asp Asp Asp Pro Asp Gly Phe Leu Gly 530 535 46 1638 DNAHomo sapiens CDS (1)...(1638) 46 atg aag aac caa gac aaa aag aac ggg gctgcc aaa caa tcc aat cca 48 Met Lys Asn Gln Asp Lys Lys Asn Gly Ala AlaLys Gln Ser Asn Pro 1 5 10 15 aaa agc agc cca gga caa ccg gaa gca ggaccc gag gga gcc cag gag 96 Lys Ser Ser Pro Gly Gln Pro Glu Ala Gly ProGlu Gly Ala Gln Glu 20 25 30 cgg ccc agc cag gcg gct cct gca gta gaa gcagaa ggt ccc ggc agc 144 Arg Pro Ser Gln Ala Ala Pro Ala Val Glu Ala GluGly Pro Gly Ser 35 40 45 agc cag gct cct cgg aag ccg gag ggt gct caa gccaga acg gct cag 192 Ser Gln Ala Pro Arg Lys Pro Glu Gly Ala Gln Ala ArgThr Ala Gln 50 55 60 tct ggg gcc ctt cgt gat gtc tct gag gag ctg agc cgccaa ctg gaa 240 Ser Gly Ala Leu Arg Asp Val Ser Glu Glu Leu Ser Arg GlnLeu Glu 65 70 75 80 gac ata ctg agc aca tac tgt gtg gac aat aac cag gggggc ccc ggc 288 Asp Ile Leu Ser Thr Tyr Cys Val Asp Asn Asn Gln Gly GlyPro Gly 85 90 95 gag gat ggg gca cag ggt gag ccg gct gaa ccc gaa gat gcagag aag 336 Glu Asp Gly Ala Gln Gly Glu Pro Ala Glu Pro Glu Asp Ala GluLys 100 105 110 tcc cgg acc tat gtg gca agg aat ggg gag cct gaa cca actcca gta 384 Ser Arg Thr Tyr Val Ala Arg Asn Gly Glu Pro Glu Pro Thr ProVal 115 120 125 gtc aat gga gag aag gaa ccc tcc aag ggg gat cca aac acagaa gag 432 Val Asn Gly Glu Lys Glu Pro Ser Lys Gly Asp Pro Asn Thr GluGlu 130 135 140 atc cgg cag agt gac gag gtc gga gac cga gac cat cga aggcca cag 480 Ile Arg Gln Ser Asp Glu Val Gly Asp Arg Asp His Arg Arg ProGln 145 150 155 160 gag aag aaa aaa gcc aag ggt ttg ggt aag gag atc acgttg ctg atg 528 Glu Lys Lys Lys Ala Lys Gly Leu Gly Lys Glu Ile Thr LeuLeu Met 165 170 175 cag aca ttg aat act ctg agt acc cca gag gag aag ctggct gct ctg 576 Gln Thr Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu AlaAla Leu 180 185 190 tgc aag aag tat gct gaa ctg ctg gag gag cac cgg aattca cag aag 624 Cys Lys Lys Tyr Ala Glu Leu Leu Glu Glu His Arg Asn SerGln Lys 195 200 205 cag atg aag ctc cta cag aaa aag cag agc cag ctg gtgcaa gag aag 672 Gln Met Lys Leu Leu Gln Lys Lys Gln Ser Gln Leu Val GlnGlu Lys 210 215 220 gac cac ctg cgc ggt gag cac agc aag gcc gtc ctg gcccgc agc aag 720 Asp His Leu Arg Gly Glu His Ser Lys Ala Val Leu Ala ArgSer Lys 225 230 235 240 ctt gag agc cta tgc cgt gag ctg cag cgg cac aaccgc tcc ctc aag 768 Leu Glu Ser Leu Cys Arg Glu Leu Gln Arg His Asn ArgSer Leu Lys 245 250 255 gaa gaa ggt gtg cag cgg gcc cgg gag gag gag gagaag cgc aag gag 816 Glu Glu Gly Val Gln Arg Ala Arg Glu Glu Glu Glu LysArg Lys Glu 260 265 270 gtg acc tcg cac ttc cag gtg aca ctg aat gac attcag ctg cag atg 864 Val Thr Ser His Phe Gln Val Thr Leu Asn Asp Ile GlnLeu Gln Met 275 280 285 gaa cag cac aat gag cgc aac tcc aag ctg cgc caagag aac atg gag 912 Glu Gln His Asn Glu Arg Asn Ser Lys Leu Arg Gln GluAsn Met Glu 290 295 300 ctg gct gag agg ctc aag aag ctg att gag cag tatgag ctg cgc gag 960 Leu Ala Glu Arg Leu Lys Lys Leu Ile Glu Gln Tyr GluLeu Arg Glu 305 310 315 320 gag cat atc gac aaa gtc ttc aaa cac aag gaccta caa cag cag ctg 1008 Glu His Ile Asp Lys Val Phe Lys His Lys Asp LeuGln Gln Gln Leu 325 330 335 gtg gat gcc aag ctc cag cag gcc cag gag atgcta aag gag gca gaa 1056 Val Asp Ala Lys Leu Gln Gln Ala Gln Glu Met LeuLys Glu Ala Glu 340 345 350 gag cgg cac cag cgg gag aag gat ttt ctc ctgaaa gag gca gta gag 1104 Glu Arg His Gln Arg Glu Lys Asp Phe Leu Leu LysGlu Ala Val Glu 355 360 365 tcc cag agg atg tgt gag ctg atg aag cag caagag acc cac ctg aag 1152 Ser Gln Arg Met Cys Glu Leu Met Lys Gln Gln GluThr His Leu Lys 370 375 380 caa cag ctt gcc cta tac aca gag aag ttt gaggag ttc cag aac aca 1200 Gln Gln Leu Ala Leu Tyr Thr Glu Lys Phe Glu GluPhe Gln Asn Thr 385 390 395 400 ctt tcc aaa agc agc gag gta ttc acc acattc aag cag gag atg gaa 1248 Leu Ser Lys Ser Ser Glu Val Phe Thr Thr PheLys Gln Glu Met Glu 405 410 415 aag atg act aag aag atc aag aag ctg gagaaa gaa acc acc atg tac 1296 Lys Met Thr Lys Lys Ile Lys Lys Leu Glu LysGlu Thr Thr Met Tyr 420 425 430 cgg tcc cgg tgg gag agc agc aac aag gccctg ctt gag atg gct gag 1344 Arg Ser Arg Trp Glu Ser Ser Asn Lys Ala LeuLeu Glu Met Ala Glu 435 440 445 gag aaa aca gtc cgg gat aaa gaa ctg gagggc ctg cag gta aaa atc 1392 Glu Lys Thr Val Arg Asp Lys Glu Leu Glu GlyLeu Gln Val Lys Ile 450 455 460 caa cgg ctg gag aag ctg tgc cgg gca ctgcag aca gag cgc aat gac 1440 Gln Arg Leu Glu Lys Leu Cys Arg Ala Leu GlnThr Glu Arg Asn Asp 465 470 475 480 ctg aac aag agg gta cag gac ctg agtgct ggt ggc cag ggc tcc ctc 1488 Leu Asn Lys Arg Val Gln Asp Leu Ser AlaGly Gly Gln Gly Ser Leu 485 490 495 act gac agt ggc cct gag agg agg ccagag ggg cct ggg gct caa gca 1536 Thr Asp Ser Gly Pro Glu Arg Arg Pro GluGly Pro Gly Ala Gln Ala 500 505 510 ccc agc tcc ccc agg gtc aca gaa gcgcct tgc tac cca gga gca ccg 1584 Pro Ser Ser Pro Arg Val Thr Glu Ala ProCys Tyr Pro Gly Ala Pro 515 520 525 agc aca gaa gca tca ggc cag act gggcct caa gag ccc acc tcc gcc 1632 Ser Thr Glu Ala Ser Gly Gln Thr Gly ProGln Glu Pro Thr Ser Ala 530 535 540 agg gcc 1638 Arg Ala 545 47 550 PRTOryctolagus cuniculus 47 Met Ala Gly Pro Pro Ala Leu Pro Pro Pro Glu ThrAla Ala Ala Ala 1 5 10 15 Thr Thr Ala Ala Ala Ala Ala Ser Ser Ser AlaAla Ser Pro His Tyr 20 25 30 Gln Glu Trp Ile Leu Asp Thr Ile Asp Ser LeuArg Ser Arg Lys Ala 35 40 45 Arg Pro Asp Leu Glu Arg Ile Cys Arg Met ValArg Arg Arg His Gly 50 55 60 Pro Glu Pro Glu Arg Thr Arg Ala Glu Leu GluLys Leu Ile Gln Gln 65 70 75 80 Arg Ala Val Leu Arg Val Ser Tyr Lys GlySer Ile Ser Tyr Arg Asn 85 90 95 Ala Ala Arg Val Gln Pro Pro Arg Arg GlyAla Thr Pro Pro Ala Pro 100 105 110 Pro Arg Ala Pro Arg Gly Gly Pro AlaAla Ala Ala Ala Pro Pro Pro 115 120 125 Thr Pro Ala Pro Pro Pro Pro ProAla Pro Val Ala Ala Ala Ala Ala 130 135 140 Pro Ala Arg Ala Pro Arg AlaAla Ala Ala Ala Ala Ala Ala Thr Ala 145 150 155 160 Pro Pro Ser Pro GlyPro Ala Gln Pro Gly Pro Arg Ala Gln Arg Ala 165 170 175 Ala Pro Leu AlaAla Pro Pro Pro Ala Pro Ala Ala Pro Pro Ala Ala 180 185 190 Ala Pro ProAla Gly Pro Arg Arg Ala Pro Pro Pro Ala Ala Ala Val 195 200 205 Ala AlaArg Glu Ser Pro Leu Pro Pro Pro Pro Gln Pro Pro Ala Pro 210 215 220 ProGln Gln Gln Gln Gln Pro Pro Pro Pro Pro Pro Pro Gln Gln Pro 225 230 235240 Gln Pro Pro Pro Glu Gly Gly Ala Ala Arg Ala Gly Gly Pro Ala Arg 245250 255 Pro Val Ser Leu Arg Glu Val Val Arg Tyr Leu Gly Gly Ser Ser Gly260 265 270 Ala Gly Gly Arg Leu Thr Arg Gly Arg Val Gln Gly Leu Leu GluGlu 275 280 285 Glu Ala Ala Ala Arg Gly Arg Leu Glu Arg Thr Arg Leu GlyAla Leu 290 295 300 Ala Leu Pro Arg Gly Asp Arg Pro Gly Arg Ala Pro ProAla Ala Ser 305 310 315 320 Ala Arg Ala Ala Arg Asn Lys Arg Ala Gly GluGlu Arg Val Leu Glu 325 330 335 Lys Glu Glu Glu Glu Glu Glu Glu Glu AspAsp Glu Asp Asp Asp Asp 340 345 350 Asp Val Val Ser Glu Gly Ser Glu ValPro Glu Ser Asp Arg Pro Ala 355 360 365 Gly Ala Gln His His Gln Leu AsnGly Gly Glu Arg Gly Pro Gln Thr 370 375 380 Ala Lys Glu Arg Ala Lys GluTrp Ser Leu Cys Gly Pro His Pro Gly 385 390 395 400 Gln Glu Glu Gly ArgGly Pro Ala Ala Gly Ser Gly Thr Arg Gln Val 405 410 415 Phe Ser Met AlaAla Leu Ser Lys Glu Gly Gly Ser Ala Ser Ser Thr 420 425 430 Thr Gly ProAsp Ser Pro Ser Pro Val Pro Leu Pro Pro Gly Lys Pro 435 440 445 Ala LeuPro Gly Ala Asp Gly Thr Pro Phe Gly Cys Pro Ala Gly Arg 450 455 460 LysGlu Lys Pro Ala Asp Pro Val Glu Trp Thr Val Met Asp Val Val 465 470 475480 Glu Tyr Phe Thr Glu Ala Gly Phe Pro Glu Gln Ala Thr Ala Phe Gln 485490 495 Glu Gln Glu Ile Asp Gly Lys Ser Leu Leu Leu Met Gln Arg Thr Asp500 505 510 Val Leu Thr Gly Leu Ser Ile Arg Leu Gly Pro Ala Leu Lys IleTyr 515 520 525 Glu His His Ile Lys Val Leu Gln Gln Gly His Phe Glu AspAsp Asp 530 535 540 Pro Glu Gly Phe Leu Gly 545 550 48 2561 DNAOryctolagus cuniculus CDS (246)...(1895) 48 ggtctgtgtg tgcgtgcgtgcgagtgagtg agtgtgtgca tatttttttt tctcttttct 60 ttctctctct tttttttttttttgcaaaga aacagcagcg ccgccgccgc tccgccgagg 120 cgctgcgccc cccgggggggggaggcggag gaggcgggca gcggcggagg gaggggagcc 180 ggggaggggg gcgccgcgctgggagggagg cagcgcgcac ggtgcagccg ggccgggcgg 240 gaggc atg gcg ggg cccccg gcc cta ccc ccg ccg gag acg gcg gcg gcc 290 Met Ala Gly Pro Pro AlaLeu Pro Pro Pro Glu Thr Ala Ala Ala 1 5 10 15 gcc acc acg gcc gcg gccgcc gcc tcg tcg tcc gcc gct tcc ccg cac 338 Ala Thr Thr Ala Ala Ala AlaAla Ser Ser Ser Ala Ala Ser Pro His 20 25 30 tac caa gag tgg att ctg gacacc atc gac tcg ctg cgc tcg cgc aag 386 Tyr Gln Glu Trp Ile Leu Asp ThrIle Asp Ser Leu Arg Ser Arg Lys 35 40 45 gcg cgg ccg gac ctg gag cgc atctgc cgg atg gtg cgg cgg cgg cac 434 Ala Arg Pro Asp Leu Glu Arg Ile CysArg Met Val Arg Arg Arg His 50 55 60 ggc ccg gag ccg gag cgc acg cgc gccgag ctc gag aaa ctg atc cag 482 Gly Pro Glu Pro Glu Arg Thr Arg Ala GluLeu Glu Lys Leu Ile Gln 65 70 75 cag cgc gcc gtg ctc cgg gtc agc tac aagggg agc atc tcg tac cgc 530 Gln Arg Ala Val Leu Arg Val Ser Tyr Lys GlySer Ile Ser Tyr Arg 80 85 90 95 aac gcg gcg cgc gtc cag ccg ccc cgg cgcgga gcc acc ccg ccg gcc 578 Asn Ala Ala Arg Val Gln Pro Pro Arg Arg GlyAla Thr Pro Pro Ala 100 105 110 ccg ccg cgc gcc ccc cgc ggg ggc ccc gccgcc gcc gcc gcg ccg ccg 626 Pro Pro Arg Ala Pro Arg Gly Gly Pro Ala AlaAla Ala Ala Pro Pro 115 120 125 ccc acg ccc gcc ccg ccg ccg ccg ccc gcgccc gtc gcc gcc gcc gcc 674 Pro Thr Pro Ala Pro Pro Pro Pro Pro Ala ProVal Ala Ala Ala Ala 130 135 140 gcc ccg gcc cgg gcg ccc cgc gcg gcc gccgcc gcc gct gcc gcc aca 722 Ala Pro Ala Arg Ala Pro Arg Ala Ala Ala AlaAla Ala Ala Ala Thr 145 150 155 gcg ccc ccc tcg ccc ggc ccc gcg cag ccgggc ccc cgc gcg cag cgg 770 Ala Pro Pro Ser Pro Gly Pro Ala Gln Pro GlyPro Arg Ala Gln Arg 160 165 170 175 gcc gcg ccc ctg gcc gcg ccg ccg cccgcg ccc gcc gct ccc ccg gcg 818 Ala Ala Pro Leu Ala Ala Pro Pro Pro AlaPro Ala Ala Pro Pro Ala 180 185 190 gcg gcg ccc ccg gcc ggc ccg cgc cgcgcc ccc ccg ccc gcc gcc gcc 866 Ala Ala Pro Pro Ala Gly Pro Arg Arg AlaPro Pro Pro Ala Ala Ala 195 200 205 gtc gcc gcc cgg gag tcg ccg ctg ccgccg ccg cca cag ccg ccg gcg 914 Val Ala Ala Arg Glu Ser Pro Leu Pro ProPro Pro Gln Pro Pro Ala 210 215 220 ccg cca cag cag cag cag cag ccg ccgccg cca ccg ccg ccg cag cag 962 Pro Pro Gln Gln Gln Gln Gln Pro Pro ProPro Pro Pro Pro Gln Gln 225 230 235 cca cag ccg ccg ccg gag ggg ggc gcggcg cgg gcc ggc ggc ccg gcg 1010 Pro Gln Pro Pro Pro Glu Gly Gly Ala AlaArg Ala Gly Gly Pro Ala 240 245 250 255 cgg ccc gtg agc ctg cgg gaa gtcgtg cgc tac ctc ggg ggt agc agc 1058 Arg Pro Val Ser Leu Arg Glu Val ValArg Tyr Leu Gly Gly Ser Ser 260 265 270 ggc gct ggc ggc cgc ctg acc cgcggc cgc gtg cag ggt ctg ctg gaa 1106 Gly Ala Gly Gly Arg Leu Thr Arg GlyArg Val Gln Gly Leu Leu Glu 275 280 285 gag gag gcg gcg gcg cgg ggc cgcctg gag cgc acc cgt ctc gga gcg 1154 Glu Glu Ala Ala Ala Arg Gly Arg LeuGlu Arg Thr Arg Leu Gly Ala 290 295 300 ctt gcg ctg ccc cgc ggg gac aggccc gga cgg gcg cca ccg gcc gcc 1202 Leu Ala Leu Pro Arg Gly Asp Arg ProGly Arg Ala Pro Pro Ala Ala 305 310 315 agc gcc cgc gcg gcg cgg aac aagaga gct ggc gag gag cga gtg ctt 1250 Ser Ala Arg Ala Ala Arg Asn Lys ArgAla Gly Glu Glu Arg Val Leu 320 325 330 335 gaa aag gag gag gag gag gaggag gag gaa gac gac gag gac gac gac 1298 Glu Lys Glu Glu Glu Glu Glu GluGlu Glu Asp Asp Glu Asp Asp Asp 340 345 350 gac gac gtc gtg tcc gag ggctcg gag gtg ccc gag agc gat cgt ccc 1346 Asp Asp Val Val Ser Glu Gly SerGlu Val Pro Glu Ser Asp Arg Pro 355 360 365 gcg ggt gcg cag cat cac cagctg aat ggc ggc gag cgc ggc ccg cag 1394 Ala Gly Ala Gln His His Gln LeuAsn Gly Gly Glu Arg Gly Pro Gln 370 375 380 acc gcc aag gag cgg gcc aaggag tgg tcg ctg tgt ggc ccc cac cct 1442 Thr Ala Lys Glu Arg Ala Lys GluTrp Ser Leu Cys Gly Pro His Pro 385 390 395 ggc cag gag gaa ggg cgg gggccg gcc gcg ggc agt ggc acc cgc cag 1490 Gly Gln Glu Glu Gly Arg Gly ProAla Ala Gly Ser Gly Thr Arg Gln 400 405 410 415 gtg ttc tcc atg gcg gccttg agt aag gag ggg gga tca gcc tct tcg 1538 Val Phe Ser Met Ala Ala LeuSer Lys Glu Gly Gly Ser Ala Ser Ser 420 425 430 acc acc ggg cct gac tccccg tcc ccg gtg cct ttg ccc ccc ggg aag 1586 Thr Thr Gly Pro Asp Ser ProSer Pro Val Pro Leu Pro Pro Gly Lys 435 440 445 cca gcc ctc cca gga gccgat ggg acc ccc ttt ggc tgc cct gcc ggg 1634 Pro Ala Leu Pro Gly Ala AspGly Thr Pro Phe Gly Cys Pro Ala Gly 450 455 460 cgc aaa gag aag ccg gcagac ccc gtg gag tgg aca gtc atg gac gtc 1682 Arg Lys Glu Lys Pro Ala AspPro Val Glu Trp Thr Val Met Asp Val 465 470 475 gtg gag tac ttc acc gaggcg ggc ttc cct gag caa gcc acg gct ttc 1730 Val Glu Tyr Phe Thr Glu AlaGly Phe Pro Glu Gln Ala Thr Ala Phe 480 485 490 495 cag gag cag gag atcgac ggc aag tcc ctg ctg ctc atg cag cgc acc 1778 Gln Glu Gln Glu Ile AspGly Lys Ser Leu Leu Leu Met Gln Arg Thr 500 505 510 gat gtc ctc acc ggcctg tcc atc cgc ctg ggg cca gcg ttg aaa atc 1826 Asp Val Leu Thr Gly LeuSer Ile Arg Leu Gly Pro Ala Leu Lys Ile 515 520 525 tat gag cac cat atcaag gtg ctg cag cag ggt cac ttc gag gac gat 1874 Tyr Glu His His Ile LysVal Leu Gln Gln Gly His Phe Glu Asp Asp 530 535 540 gac ccg gaa ggc ttcctg gga tgagcacaga gccgccgcgc cccttgtccc 1925 Asp Pro Glu Gly Phe LeuGly 545 550 cacccccacc ccgcctggac ccattcctgc ctccatgtca cccaaggtgtcccagaggcc 1985 aggagctgga ctgggcaggc gaggggtgcg gacctaccct gattctggtagggggcgggg 2045 ccttgctgtg ctcattgcta cccccccacc ccgtgtgtgt ctctgcacctgcccccagca 2105 cacccctccc ggagcctgga tgtcgcctgg gactctggcc tgctcattttgcccccagat 2165 cagccccctc cctccctcct gtcccaggac attttttaaa agaaaaaaaggaaaaaaaaa 2225 aattggggag ggggctggga aggtgcccca agatcctcct cggcccaaccaggtgtttat 2285 tcctatatat atatatatat gttttgttct gcctgttttt cgttttttggtgcgtggcct 2345 ttcttccctc ccaccaccac tcatggcccc agccctgctc gccctgtcggcgggagcagc 2405 tgggaatggg aggagggtgg gaccttgggt ctgtctccca ccctctctcccgttggttct 2465 gttgtcgctc cagctggctg tattgctttt taatattgca ccgaagggttgttttttttt 2525 ttttaaataa aattttaaaa aaaggaaaaa aaaaaa 2561 49 12619DNA Homo sapiens 49 aagctttata aagatttaac tacctaataa ggtagagaagtaatttatgt gcccactaaa 60 aaatactcaa tttctgaatg ttcgtccaaa attaacttgtcagatcatta aatcattgac 120 tagaaacacg ttgagtacct attatgtact aggcacttagatcattgtga gacaataaaa 180 aatactgcat tagaaaagga catttttcac atcttaaatgcaataagcat tatttggctg 240 gcagttaatt acatttaaca cattaaacat atagagcaaaattctgagca atcaaaataa 300 ttataccctt gagcaatcga ttatttaaat ttctttcactattcccttaa gctgatttct 360 actctgggat tctttcatag ttctcaaata agaaaataaaaaatttccta aataaggcaa 420 tacaaaagaa tagaaatgta agagaagaga tatattagctcttgaatccc tgtttccatt 480 tgctgtcaat agtgcctcta atgttcgatt ttctcttcaaagaaaaatct tgatttaaaa 540 ggaagaaaaa gtacaatcac ctttaacagc taaagtatactgattagcat ctactaaagt 600 tagcaaagac tgaaactgaa aaaaaattgt aaaatctttattctaagtta tataacgcca 660 ttcaccatag taatgatttt atactttggt atatggctttttaaaataaa tattgccaac 720 aggtaaaaat ttttcctttg ctgtcttaag gcattcctaagagaattttt accagtgtgt 780 gttcataact tgaatgttaa tttaaacaat gttacttctatcacctaaat gatatactta 840 tagaagagtg gtttaattgg gaacagaaaa acaccacattgcttcttccc aagaaaaagg 900 gatgtattcc attctcgagg tctctctccc actctctatttatatataat atactgcata 960 gataaatata cacacattat atatgtattt ttttgaacttaaagaagact ggacatatgt 1020 atttacatgt atatatccaa caaatattta attttgagatctctctccct cttctgattt 1080 attattctca gtatgaattc tcaaactgta cggtctttcacatttcattc attcatcaag 1140 catgtatcga gtcccttctg catgcttagc tttttgtcatatggaaggaa gatacaaaag 1200 aaaaactgtt tctgcccttc agaatctttc catctcttctaggaaggaga taaaacacca 1260 tatatcatta agaaatttat aagactagtc ccaaaaccaatggtacaagc aacatgcatt 1320 ttacatttat gtagaatttt agagcttgga aacactttcgtgatatataa tcctaagaac 1380 aatcttgtaa agtgcacatt attagctcca tttcagtgatgaggaatctg agacagaatt 1440 ttaagtgaca tgtctcgttc aaacattatg agtggaagagtcaacactta agcctgagtt 1500 ttctgattct aagcctagtg ctcttttcaa cacagcactggaaaccaaag attgtggtac 1560 acaacaaggc aacagccagt cttcttgctc gaggtccaactaaactggac ccataccgag 1620 cagtgtccag ccaaatgtcc aaattaattt tatcctgcaaatatttgttc ttcagtgtaa 1680 tacacacagc acaactacca tttccttcgt cttagtgcctttatctccta cattccagaa 1740 atggggatgt caaatatttt tttaaatctg gcctagatggaatcatataa atctcaaatc 1800 ataatataaa tcttaaaggt ctggtttcca ccaatccttccacattttgt tttcccccag 1860 cactagagag cctaacctac cctcacccct ttcgagcattcttgctccaa acgaccacct 1920 attttaagat gtcaatgacc ctttcccaaa ttctacaaattcaccccagt tttgccaccc 1980 gaccccagcg cctgcccgga cacgttcccc tccctcccaatagatttgat accgagttca 2040 ggttctgcag atcccgttgc gatgctgtca cacagcactgacagataaga tttgaccttt 2100 cgactccgtc cttggggact tcccgctggc caagaagggtagttccaatc ccaggaaacg 2160 ggcttcctgc tcaggaacgc agcctctagc agcgcacagtctgaggcaat gtctccggca 2220 attagaacga tgctgggcgc ccgggtgtgc atcactctgcctcatactcc taccaactgc 2280 agggcactcg gtccggcagc cagtccatcc cacccacacccaagtcccag ccagccggac 2340 cttacgcagg accccgatga taggtcgttg acggctgcagcaaaagccaa ggccacctgc 2400 cgctgctgcc catccccgcc aatctgagac cccctagactggaccgcaga aaagcgtttc 2460 tatgggaacc cccccaccga gaatcacgtg acgcaatcggacgaccaatc gcttcttacc 2520 tctgcccgcg gtccagcttt tggccctccc tctcgcccccgcctccttcg cccagccccg 2580 ccccttgcct gcggagagcc cgcgcctgcg cgctgtgtcctgcgcgctcc ttccctcgcg 2640 cgcgctctcc gtggaagagc aggggcagcg tgggaggcgccaagggagcg cgaacctgag 2700 gaggaagaaa cggggctagc gcgcaggccc agaacggtccgagccgcggc agtcggcgac 2760 gcctcagagc ggaagaggga agtgaatcag gcgccgggtagtgggttgct gggctgggct 2820 tgctgaggta gaggcagcgc caagaagagg cctttgccgctggtcgggat tgggatgtcg 2880 aagaacacag tgtcgtcggc ccgcttccgg aaggtggacgtggatgaata tgacgagaac 2940 aagttcgtgg acgaagaaga tgggggcgac ggccaggccgggcccgacga gggcgaggtg 3000 gactcctgcc tgcggcaata tccttgcatt caccgccctccccaccccag cccagcccag 3060 cccgcccttc tcctgggacc cgggagcctg caggatccgcggggcaccgg cgcggagctg 3120 cctctcaacc tgcggcttaa cctgtctctt tgggatcgcccgctctgaga gggcaagggg 3180 gaagcccccg tttcctaccc agtcggcagg agacgcgagggtcccactct tggaagcctg 3240 ccctaccccg cgcgccttcc acgcccccag attcctcaggttgcacccga gtgcctgcct 3300 gcctcgggaa ctggtcccgc cgcccgcgcc ctcgcggcgctggggaaggc ggccccggct 3360 ggtggggaag gctggtgccg accgccttag tttttcttcctagaactctg atttcctggg 3420 gtcacattag ctccagaaat ttctgattgt ggggaacctgcatctttcct tagtggtttt 3480 gttttttggt tgtgtttttg ttattggtag cgttaaggtagtttattgct taccgggggg 3540 ccgggggaga tgggactgtt cgaaaattga gggtccctgtgctttcagcc cattggcctt 3600 tttaaaaaaa aaaaaaaaag aagaagaaga aggggatttggcaaaatata cattgtacag 3660 aatttgttaa ctgggggagg ggaatgaata caaaaaatacaaaactccta gaaggaagct 3720 tggagccttt tacctgctaa gaaaaggaca atagaaaaaacaacggggaa tgcgtgtgga 3780 gaatccttgg aaatatttaa aataaacccc aatgaataagatagaagatg agtcattcgt 3840 ataaagcaga atcatttttg taatcctaaa attgtttccattttagttaa aatatggcag 3900 tcagttcccg gtttctgttt ttgcatattt gaatattcataactttggct tcgcatttgc 3960 attacatctt ttttagaaaa atgtaaatgt tgcaaaaaaaccgaagctgt agttttagaa 4020 aatctcagac actgaatttg tatgcatttc taattcttgggtgtattcat aaggaagact 4080 ctcaacaatg tcctgttata gtggggaaat atgagagtgaaaatatttaa tggcaacaat 4140 atcctttttt aaaggcacct aaatagagca ttagacatttatcaatatat agatagtgct 4200 ttgcccaact ttcacaatta attagctgtt gctcttttgcattatttaaa tacttaagtg 4260 cttggagtta taaaaaatga gctaatctac atcaggcatgcttctctaga aatccctgca 4320 gccttgaaaa taacagcttg tcaaccagag attttgtgtaagaacttttt ctttagaaaa 4380 taaatggtga acatgcttcc taaaaacatt atttgtgatgggataagatg gtgttttatg 4440 aaaccccagt gtattttagg taatttgtgg tgacttttaaaaggtactgc tgtatccata 4500 tcagtggatc tgctttttga tcagttcatc ttaaaatataaagatactgt ctcttcttac 4560 cgttacatac agccaggaaa gacagcccta gtggtggggtactagagttg gaggaacaag 4620 tgaactctgt ggttttcctt ttaggggaat gtttgtacattctgacagtc tgattggcct 4680 tctgtttctc atgcttgcta actcactagt gctttcaaagagagcctgaa tttaataggt 4740 atggtctaac acagtttgaa taacctttgt gaaatatgagagaaaatatc taaagcaaaa 4800 aattaagctg ccacctaagg gacatatgaa ttattacatcttctgtgatg cctcttttca 4860 tcaatattga gagattgcta atgtgtatca ttcagattgctaatctgcca gcatgttcta 4920 ccagcatttc agataataca gaatatggtt ctagcaaaagtttggtcttt attttttcaa 4980 ttagaatcac aggaaaagac atattttggt tgataataggttatttcatt tgggggacta 5040 ataattctga tatatatttt aggatttctt taacaccactctaggtaatg tttgcatatg 5100 tatctcactg ggaaatgaaa gactatcaag gtgttcacttgatagttaga accaagggtg 5160 aaacagtctt tgctttatta aaaaaaagtc taatgttctattttgctttt gatattttgc 5220 ctttgattaa catcctggaa accaacacat tgaatttccagtattgaaca tagtgaccaa 5280 agtaattttc tttttatatg taaatcaagt cataaagaaccagtggttat aatgctttct 5340 gggggccatc ctttgctgtt acacccttaa cttccatcacaggaaacatg acagctgccc 5400 tacaggcagc tctgaagaac ccccctatca acaccaagagtcaggcagtg aaggtgagtc 5460 gcagactaca acacagtgat ctctgctgat atcttattcttagtaaaatc cttgcagtgc 5520 aaaaaaaaat caatatttta actgtttgct atctttgacaagaagagttt ataatgtagt 5580 ttgataggta aaaatttcac gtgaaaaaat agccctataatgtagttatg ataatgctgc 5640 atggtaagat acagtaagtt caaacgatag tgaaatcatttgtgtgtgtt tttagaggag 5700 accactcagg ctgaatttga gcaaaggttt gaaaaataagttaaaccttt acaaaaataa 5760 acagattgta attgcttttt aaagattttt taaaaccatacaaatactaa atacttatta 5820 tagaaagctc agacatatga gaaggttaaa aagatagtggtttgtggtcc cagcacccag 5880 agataacagt tactactttg gggccttgct gtattgttacagagttccct tttgtttttt 5940 taagaatgaa tttttaaaac gggctttttc agctatatgcaatggtacat gagctttcct 6000 tccccaataa gttaatagcc ttttttaaca cttgtatatggataagctcc agtgtataca 6060 taactaatct tttgtttata tttagactga ctttttttttcctattgtaa accactgaaa 6120 tcaatatttt ttggtaaatt tttaattgtt ctctttgagtaaattgctag cagtgaatta 6180 ctggatcaaa gaatgcactt ttttttaagg cttttggtatgcagtattgc caaattgccc 6240 ttcagaacag ttgtgcaact tacattctct gcagtcttttactaattctt aacctattta 6300 cgtatttatt taaaatgatg cccatagcat caaccccgttgtccatagct attcatacat 6360 cctaggagct tcaagaatct caattgaata gtagtaagtaataacttagg taaatgcata 6420 ataattatct aggtaacata attttttatt ggggaaaatttctttggttt ttacaagttg 6480 taaagattgt cgttgaaatt tcatttttac cgtggatgcaaagatatttt tctaaatctg 6540 gtaattgcag tctttaaacc aaagataaca gtaggtggtagaaacattct gtgaaatcct 6600 gaccagtagg aatgctggag gtatcacttt gtgttgaatggaaggagaaa cgaattgttg 6660 aaaaggtcag ttaagtgttt cctttgcttg gccggatgggtaagaaaata actgcttttg 6720 aagcaggctt ttgccaaaga aaaaagatca ttattaatgaacatcactat atttcatatc 6780 tacagtcaat tcatataaat tacagtcaat tttcttttaagacagcttgg tttattaaaa 6840 tttttaaata aaaaagtttt taagaaaaaa ttacttctgaaggataattc aaggtgaaac 6900 tgcaaatctg cctccttgtt ttgttgggaa tttttttttttttttttttt ttttgagacg 6960 gagtctcact ctatcaccca ggttggagtg cagtggtgcaatctcaactc actgcaccct 7020 ccgcctcccg ggtttaagca atcctcctgc ttcagcctcccgagtagctg ggatcacagg 7080 cacacaccac catgcctgga taatttctgt atttttagaagaaaacaggg ttttaccatt 7140 ttggccaggc tggtctcgaa ctcctgacct caggtgatctgcccatctcg gcctcccaaa 7200 gtgctgggat tacagctgtg ggccaccaca cccggccgttttgttgggat tttttttttt 7260 taagatcaag acataaattt aaatgttgtt ttaataaattgttaaattat cacattgatc 7320 tgttagcaaa tcctctcagc tctgccttca attatgttaatagtctgtca agtttcttac 7380 cacctccact gctactatgc ttaccacatc cagcctgtattattgcaatt gcctcctaat 7440 tgctctccct gcttctacct tatcccctac tcccacagcttattttctgt aacatagatg 7500 ccaaagcaat cctgttaaaa tgtgagtcag attatggcactgctcttaaa accttccaat 7560 gtcttctcat ttctctcagt aaaagccaaa ctccttacaatgcctgtagg ccttacacga 7620 tctgtcctcc cataacctct gacttactca cgtgcttttctcccaccaat ccactccaac 7680 cacattgggt ttttttctgt tcctggaaca cactgaacacacactaatag cactgttctt 7740 tcctctgtct gaaacacttt cctcagttat cccaagccttctttcacgtc cttcaggtcc 7800 ttactcaaat gtcacattca tagtgtagac tttctgaaattctaaaccct cctcatacag 7860 atatgtctaa atgttctgtt atttattgac ccaccaggaccgggcaggca gcattgtctt 7920 gaaggtgctc atctctttta aagctaatga tatagaaaaggcagttcaat ctctggacaa 7980 gaatggtgtg gatctcctaa tgaagtatat ttataaaggatttgagagcc cgtctgacaa 8040 tagcagtgct atgttactgc aatggcatga aaaggtaagttatgaattat aaatctatat 8100 gactggttct tttacaatag ggaatgacaa tgacaacctctctcacctaa ataaccattt 8160 tgatttgttg tacatttttg ttattacaaa taaaatgcatgaaaaggata gttcatattt 8220 atgtttacta gccttggtct taagagattc tgattccaacacttgtgttt attcaacaat 8280 gattattagt aattaaacat aatcttgaac tctgaattaaatcaaaactt tgtaaaagaa 8340 aataagcaat acaaatcaag aattctttca cagtgaccaaaaggtgaaaa caacacaagg 8400 atcgaatatg attcaaccat taaaaggaat gacattctgacacatgctat aacattaata 8460 aaccttgaaa acataccaag tgaaatgagc caaacacaaaagaactaata ttttataatt 8520 ttacttatat gaaataatct aggataggca aacacaaagggacagaaagt ccttagaggt 8580 tactaggaag tagggaaagc aaggaatagg gagttagtgcttaataggta cagagttcct 8640 ccttggagtg gtaaaaaagt tttggaaaca gatagtggtgatggctacag tacattgtga 8700 atataattaa tgccaatgga ttttacactt aaagatggttaaaatggcaa attttgtgtt 8760 agatatttta caactttttt aaagaattag gagtttggaggatcaagaat tcttaaatca 8820 tgtttttcta ttttcatgtg tatattttgc aatgtaagtagatgctggta catcatctgt 8880 caaaagagta taagtgattt tgagctttgg gtaaaaaactggataacatg taaatagaac 8940 cagtcataaa aatattgagt gtttgaagtg tatctgagtgaaaacacaaa cataagaaaa 9000 aagcacatag taaaacaata gttccccctt ttactctaaaatgcaccaat ttgggtagta 9060 atttatatgg caccctattc atggaacact ttctgttgccaggtaccata ctattaatgt 9120 tttatttaac ctttacaaca accctgtgga agtatataaatatctttatc atcctcaatt 9180 tacagatgaa aagctagctt taaaacccaa gccagcgtagttctagcata gcctcaagat 9240 tgcagtgaac attgattact tattatattc cacatattcttcaaaggact ttataaatat 9300 taactcattt aatcctcata aaaatggagg gaaatgcttgctattattcc tcttttgtca 9360 ctgaggaaac tgaggcatgt gtgaagtctt catttcttccaaatgtcagt caccagtttt 9420 taccaatctt cgaagtattt ctgaaatcta tctgttcaagcgtatctaat gcagctgttc 9480 acagcatctc tcccagtctg ttgccatagc ttcctgactggtttcccagt taacagtttt 9540 gcctccttca aatctgttct ccacccagcc atcaaaatgatatctttaaa atcaaaattg 9600 cccttgtcag tcacctgcag ggataaagtc aaagttcccaagtctagctt catcttccat 9660 gtcattcttc ccctcaggct atagcaatgc cagcctttttcctgaatgca ccatattgtt 9720 tcacacctcc atacatttgc tcatgatttt ctggtgttagcctgtcacct actcattctt 9780 ttaatgtgtc atttcctcca tgaagcctta gctgaaacattcctctatac tgttaatctg 9840 ggtataagcc tctccctggt gctttaatag cacctgcagcacaactctca tttcatacat 9900 tagattaaaa ttacctgttt atatgtctgt ctcctcatgctagaccagaa aatgctgtat 9960 ttgttcactt ttgtatcccc agcatctagc acagtactcagtatacaaag gtattccata 10020 aatatttttt gaacagaaag aaaccagagc tcagattcctaatacttgat cattactctc 10080 tatttttcaa attagagtca gagttaaagt ttctaagttcttagctatta aacaatacct 10140 tctttctttg ggagaaaaaa aatctgacaa aggctgactaatcgaagtgg aagttgggat 10200 ggttgatccc agtttgaatt ttcttctgac tatgtggtgagaatgagaaa tgcagaatgt 10260 ccacctgttt tgagcaggaa cactatgctg cagatttttttttttttttt tttttttttt 10320 ttttgagacg gagtcttgct ctgtcgccca ggctggagtgcagtggcgca atctcggctc 10380 actgcaagct ccgcctcctg ggttcacacc attgtcctgcctcagcctcc cgagtagctg 10440 ggactacagg cacccgccac cacgcccggc taattttttgtatttttagt agagacgggg 10500 tttcaccatg ttagccagga tggtcttgat ctcctgacctcgtgatccgc cggcctcggc 10560 ctcccaaagt gctgggatta caggcgtgag ccaccgcgcccggcctatgc tgcagatttt 10620 ttaaaacatt atttagaatt aatgtactaa aatgtaaactagtatctcac tagaatgtaa 10680 cttcatgagg gcagggactt tcaaggtttt gtttattactgtaacctcag tgccaagaac 10740 agtacctggt gcataattgg tgctcaagaa tttattatttgttaactaat aaattcaggg 10800 tctatagcag tgcccattcc ttctttaaga aaaatgttttaccaaatatg agaattgacc 10860 ttttattatt ctgtcaacat ttacatcctg gtttgtttttaggcacttgc tgctggagga 10920 gtagggtcca ttgttcgtgt cttgactgca agaaaaactgtgtagtctgg caggaagtgg 10980 attatctgcc tcgggagtgg gaattgctgg tacaaagaccaaaacaacca aatgccaccg 11040 ctgccctgtg ggtagcatct gtttctctca gctttgccttcttgcttttt catatctgta 11100 aagaaaaaaa ttacatatca gttgtccttt aatgaaaattgggataatat agaagaaatt 11160 gtgttaaaat agaagtgttt catcctttca aaaccatttcagtgatgttt ataccaatct 11220 gtatatagta taatttacat tcaagtttaa ttgtgcaacttttaacccct gttggctggt 11280 tttttgttct gttttgtttt gtattatttt taactaatactgagagattt ggtcagaatt 11340 tgaggccagt ttcctagctc attgctagtc agggaaatgatatttataaa aaatatgaga 11400 gactggcagc tattaacatt gcaaaactgg accatatttcccttatttaa taagcaaaat 11460 atgtttttgg aataagtggt gggtgaatac cactgctaagttatagcttt gtttttgctt 11520 gcctcctgat tatctgtact gtgggtttaa gtatgctactttctctcagc atccaataat 11580 catggcccct caatttattt gtggtcaccc agggttcagagcaagaagtc ttgctttata 11640 caaatgtatc cataaaatat cagagcttgt tgggcatgaacatcaaactt ttgttccact 11700 aatatggctc tgtttggaaa aaactgcaaa tcagaaagaatgatttgcag aaagaaagaa 11760 aaactatggt gtaatttaaa ctctgggcag cctctgaatgaaatgctact ttctttagaa 11820 atataatagc tgccttagac attatgaggt atacaactagtatttaagat accatttaat 11880 atgccccgta aatgtcttca gtgttcttca gggtagttgggatctcaaaa gatttggttc 11940 agatccaaac aaatacacat tctgtgtttt agctcagtgttttctaaaaa aagaaactgc 12000 cacacagcaa aaaattgttt actttgttgg acaaaccaaatcagttctca aaaaatgacc 12060 ggtgcttata aaaagttata aatatcgagt agctctaaaacaaaccacct gaccaagagg 12120 gaagtgagct tgtgcttagt atttacattg gatgccagttttgtaatcac tgacttatgt 12180 gcaaactggt gcagaaattc tataaactct ttgctgtttttgatacctgc tttttgtttc 12240 attttgtttt gttttgtaaa aatgataaaa cttcagaaaataaaatgtca gtgttgaata 12300 atttattttt ctctgacact ttaacaatta tgaatgtatggttaattaag aggaaaggtt 12360 ttctgcttct accaccaagt actgtactct taacaagaacagtttggtag ggtttttata 12420 agactatata gatataagat gatagagaag agagtcatgaatgatgtcag agcactactg 12480 aagcctttgg agtgattcca tagccttctg gatggcagctgaatacctat atgtagtatc 12540 actgcccaaa gacctagact agaaagtgca aagtagcttagcagctgcag tcattcactc 12600 ccagcctcca aaattctct 12619 50 12425 DNA Homosapiens 50 gatccctctc caggtggaag ctcccttcat accaaagttt aaaggccctggggatacgag 60 taactttgac gactatgagg aagaagaaat ccgggtctcc atcaatgagaagtgtggcaa 120 ggagttttct gagttttagg ggcatgcctg tgcccccatg ggttttcttttttctttttt 180 cttttttttg gtcggggggg tgggagggtt ggattgaaca gccagagggccccagagttc 240 cttgcatcta atttcacccc caccccaccc tccagggtta gggggagcaggaagcccaga 300 taatcagagg gacagaaaca ccagctgctc cccctcatcc ccttcaccctcctgccccct 360 ctcccacttt tcccttcctc tttccccaca gccccccagc ccctcagccctcccagccca 420 cttctgcctg ttttaaacga gtttctcaac tccagtcaga ccaggtcttgctggtgtatc 480 cagggacagg gtatggaaag aggggctcac gcttaactcc agcccccacccacaccccca 540 tcccacccaa ccacaggccc cacttgctaa gggcaaatga acgaagcgccaaccttcctt 600 tcggagtaat cctgcctggg aaggagagat ttttagtgac atgttcagtgggttgcttgc 660 tagaattttt ttaaaaaaac aacaatttaa aatcttattt aagttccaccagtgcctccc 720 tccctccttc ctctactccc acccctccca tgtcccccca ttcctcaaatccattttaaa 780 gagaagcaga ctgactttgg aaagggaggc gctggggttt gaacctccccgctgctaatc 840 tcccctgggc ccctccccgg ggaatcctct ctgccaatcc tgcgagggtctaggcccctt 900 taggaagcct ccgctctctt tttccccaac agacctgtct tcacccttgggctttgaaag 960 ccagacaaag cagctgcccc tctccctgcc aaagaggagt catcccccaaaaagacagag 1020 ggggagcccc aagcccaagt ctttcctccc agcagcgttt ccccccaactccttaatttt 1080 attctccgct agattttaac gtccagcctt ccctcagctg agtggggagggcatccctgc 1140 aaaagggaac agaagaggcc aagtcccccc aagccacggc ccggggttcaaggctagagc 1200 tgctggggag gggctgcctg ttttactcac ccaccagctt ccgcctcccccatcctgggc 1260 gcccctcctc cagcttagct gtcagctgtc catcacctct cccccactttctcatttgtg 1320 cttttttctc tcgtaataga aaagtgggga gccgctgggg agccaccccattcatccccg 1380 tatttccccc tctcataact tctccccatc ccaggaggag ttctcaggcctggggtgggg 1440 ccccgggtgg gtgcgggggc gattcaacct gtgtgctgcg aaggacgagacttcctcttg 1500 aacagtgtgc tgttgtaaac atatttgaaa actattacca ataaagttttgtttaaaaaa 1560 aaagtgtcgc tggtgttctc gacttcgatc acccacccac acacccccagggggttggaa 1620 agggaatttc ggaccccagc gtgcaggccg atcaggtcct ggcttgaagtccttgtaacc 1680 agggtttagc tgaaattccg gcactccttc ggccccgcag gagaaacgagcgtcaaactg 1740 ccctttgacc ccagattcgg ggtccccaaa tctgcggcgc gccccctcggcgtccagccc 1800 gggaccgaga gggcgctcta gggaggcgct ggggctggcg cgccaggaggccgagcggcg 1860 gcgggggcgg ccctggcagg gggagtagaa gggggagagg gtgcgcgccccccttcccgc 1920 atcctcagcg ccgggccagg cgcgcctgag ggacgcgggg gcggcggcagcaggagggtc 1980 cccgcagcac cctgcgagcg cggcagcccc ggcccgcggg cggcgagttcccggtaagtg 2040 cggtcccgag agcggagcgc gctggagagg cgtggagagg ggggctgggcgccggggacg 2100 tctgggtccc gcgcccaatg gctggagggc ggccgagcgc cgcccgcccgccctgcccgc 2160 cccctctccc ctccccccgg cactcccctc cccctccccc gcccgccgctttcccccgcc 2220 cccgccccgg cgccaactcc gcggcgcctc cttaaaaagc gcgcgggagttgtaaggggg 2280 ggccggagcg agccggagtg agcgagagcg cagggtaaag ggggcgggcggggggcccgg 2340 gctccacctt aaaagcgggc gcgtgggggt gggagggagg aaggcgggcggcggggagga 2400 gggagggagg gaaggaaggg gggccggagt gtcccgggcg cagggcgcgcgtgcggcggc 2460 ggcggcggcg gggaggggcc ggccgcgccg cgctcccctc ctccccctcgcatccccggc 2520 cccgcgcgcg cccagcagaa gcgggtctgt gtgtgcgtgc gtgcgagtgagtgagtgtgt 2580 gcatattttt ttctctcttt tctttctctc tcactgtttt ttcctctctctctctctccc 2640 tctctctctc tttttttttt tttttttttt gcaaagaaac agcagcgccgccgccgctcc 2700 gccgaggcgc tgcgcccccc ggggggggag gcggaggagg cgggcagcggcggagggagg 2760 ggagccgggg aggggggcgc cgcgctggga gggaggcagc gcgcacggtgcagccgggcc 2820 gggcgggagg catggcgggg cccccggccc tacccccgcc ggagacggcggcggccgcca 2880 ccacggcggc cgccgcctcg tcgtccgccg cttccccgca ctaccaagagtggatcctgg 2940 acaccatcga ctcgctgcgc tcgcgcaagg cgcggccgga cctggagcgcatctgccgga 3000 tggtgcggcg gcggcacggc ccggagccgg agcgcacgcg cgccgagctcgagaaactga 3060 tccagcagcg cgccgtgctc cgggtcagct acaaggggag catctcgtaccgcaacgcgg 3120 cgcgcgtcca gccgccccgg cgcggagcca ccccgccggc cccgccgcgcgccccccgcg 3180 gggcccccgc cgccgccgcc gccgccgcgc cgccgcccac gcccgccccgccgccaccgc 3240 ccgcgcccgt cgccgccgcc gccccggccc gggcgccccg cgcggccgccgccgccgcca 3300 cagcgccccc ctcgcctggc cccgcgcagc cgggcccccg cgcgcagcgggccgcgcccc 3360 tggccgcgcc gccgcccgcg ccagccgctc ccccggcggt ggcgcccccggccggcccgc 3420 gccgcgcccc cccgcccgcc gtcgccgccc gggagccgcc gctgccgccgccgccacagc 3480 cgccggcgcc gccacagcag cagcagccgc cgccgccgca gccacagccgccgccggagg 3540 ggggcgcggt gcgggccggc ggcgcggcgc ggcccgtgag cctgcgggaagtcgtgcgct 3600 acctcggggg cagcggcggc gccggcggtc gcctaacccg cggccgcgtgcaggggctgc 3660 tggaggagga ggcggcggct cgaggccgtc tggagcgcac ccgtctcggagcgcttgcgc 3720 tgccccgcgg ggacaggccc ggacgggcgc cgccggccgc cagcgcccgcccgtctcgca 3780 gcaaggtgag cgcgccgggg agcgggggcg ccgcgcggtg ggcaggtgcgggcgaagttg 3840 gtggcggggg cgcgagtccc gggaggaact gggtggcggg tggctggggctttgcgcgcg 3900 tttcctgcgg gctcggtgcg tggtgacctt ggcaagtgat tgaatctcccggagcctcag 3960 tttcctccgc tgtaaacgcg gtttaataac agtagcgacc ccttggggttgttgagcgag 4020 tttagtaaga tttggttgtc gagggcttta gttaacacag agcctggcacggagtgaatg 4080 cgtaaaagtt agtccgtatt gttcttaaag gtggaatcgg ttcctcctccccaccgcccg 4140 gacgccacag tcagggtctg ggattagaac agctactaat tttgcatgcttctctcctcg 4200 gctccagaga ggtggagaag agcgagtact tgagaaagaa gaggaagaagatgatgatga 4260 agatgaagat gaagaagatg atgtgtcaga gggctctgaa gtgcccgagagtgaccgtcc 4320 tgcaggtgcc cagcaccacc agcttaacgg cgagcgggga cctcagagtgccaaggagag 4380 ggtcaaggag tggaccccct gcggaccgca ccagggccag gatgaagggcgggggccagc 4440 cccgggcagc ggcacccgcc aggtgttctc catggcagcc atgaacaaggaagggggaac 4500 aggtaaggat ccctctgggt ggggaagagt gctaggtgga gaggaactcagcccgaagac 4560 aaagccaaag acaggtgttt ttttccttcc cagcttctgt tgccaccgggccagactccc 4620 cgtcccccgt gcctttgccc ccaggcaaac cagccctacc tggggccgacgggaccccct 4680 ttggctgtcc gtaagttggg gtattggaga catgggggtg ctgctcaggtgtgtggtaca 4740 gccagagaga catccgtgtt cactggtgtc tgtttgtttt gatgcagtcccgggcgcaaa 4800 gagaagccat ctgatcccgt cgagtggacc gtgatggatg tcgtcgaatattttactgag 4860 gctggattcc cggagcaggc gacagctttc caagagcagg tgagtttccagcccaggact 4920 acacactgac agacacagag ggcctccctg ggatgtgccc tgatcccggctttctctgtt 4980 cctgtcccac ccaggaaatt gatggcaaat ctttgctgct catgcagcgcacagatgtgc 5040 tcaccggcct gtccatccgc ctcgggccag ccctgaaaat ctacgagcaccacatcaagg 5100 tgcttcagca aggccacttt gaggatgatg accccgatgg cttcttaggctgagcgccca 5160 gcctcacccc tgccccagcc cattccggcc cccatctcac ccaagatcccccagagtcca 5220 ggagctggac ggggacaccc tcagccctca taacagattc caaggagagggcaccctctt 5280 gtccttatct ttgccccttg tgtctgtctc acacacatct gctcctcagcacgtcggtgt 5340 ggggagggga ttgctcctta aaccccaggt ggctgaccct ccccacccagtccaggacat 5400 tttaggaaaa aaaaaatgaa atgtgggggg cttctcatct ccccaagatcctcttccgtt 5460 cagccagatg tttcctgtat aaatgtttgg atctgcctgt ttattttggtgggtggtctt 5520 tcctccctcc cctaccaccc atgcccccct tctcagtctg cccctggcctccagccccta 5580 ggggactagc tgggttgggg ttcctcgggc cttttctctc ctccctttttctttctgttg 5640 attgtcgctc cagctggctg tattgctttt taatattgca ccgaaggttttttaaataaa 5700 attttaaaaa aagaaaaagg gaaaaaaaag ccacggagtc cattttatgaatggggtggg 5760 gagagggcac taaagagcct cctaagagag cctcaggtta ggacagaattgtttggggag 5820 ggagaaaaac agaaacaatg aattatagct gcctcacagc catgtataacaataattgct 5880 ccaggaaggt gggaatattt gctttttttt cttctgtaat ctcaccgtgtccgtgtccag 5940 aacagagcta ggcacacagc aggtgctcaa tttttgtttt tcgtttagacaggtttcatt 6000 ctttcaccca ggctggagtg cagtggtgct atcatagctc attgtagcctcaaactcctg 6060 ggctgaagtg atcctcccac ctcagcctcc tgagtagctg ggactacaggtgcactctgc 6120 catgccgggc taacttttaa aaatttttgt ccgggcacag tggctcatgcctgtaatccc 6180 agcactttgg gaggccgagg tgggtggatc atgaggtcag gagttcaagatcagcctggc 6240 caagatgatg aaaccctgtc tctactaaaa atataaaaaa aaattagctgggcgtggtgg 6300 tgggtgcctg taatcctagc tattcaggag gctgaggcag aggattgcttacacctggga 6360 ggcggagggt gcagtgagcc aagatcgtgc cactgcactc cagcctgggtgacaaagtga 6420 gactctgtct caaaaaaaaa tctttgtgtg tgtgtggaga tgagggtatgcactttgttg 6480 gccaggttgg cctcgaactc ccagccaagc aattctgcct gggattacaagcgtgagcca 6540 ccatgcctgg cctcaaatat tgttgaatgg ctagcagtta agtccttgggtttataagca 6600 tttcctcaac tgtcctccca agtccccata agacaaaaaa ctcataaaatcccaccttac 6660 agaagaggca gctggcccgg cacagagatg ctgtctgccc cgggtcacacagggtggcat 6720 ctgacaccct gtctgagttc ttcactcaga gtctttaaat ataattagcgtatttgacat 6780 aatgtacatt aaaaactata aacctgtcag cctttgtcta ctgcaaagaatccactacaa 6840 atattggggc agggatctgt tcttggacca tagtagtgtc tccagacctcatggtcctct 6900 tcattaaaac aacagaaaat tccttctggg ccatcagatg agaccatgagatagaagatt 6960 tccaagtgaa gattttgttt caagacagag tcttgctctg tcactcaggctagagtgtac 7020 tggtgcaatc ataactgtgg tgacagcctc gaacttttgg gtacaagtgattctcatgcc 7080 tcagacaaca cccaactaat attttggttt ttgtatagac agggtcttgctatgtggctt 7140 aggctggtct tgaactcctg gcctcaagca gtcctcccgc ttcagcctcctaaagtgtca 7200 ggattacaga catgagccac caagtccagc ctgaagattt ttaaaaattattgttagtag 7260 tagtcgccag agttactaca tccaaagtcc ctactaagtt ctaagtagtccctactaagt 7320 tctaaggcag tttctcaact cattagagtt gttttttgtt tttaaagaaaaaaagaggct 7380 gggcacttta ggagaccgac acgggaggat cgcttgagtc caggagtttgagaccaacct 7440 gggcaacatg ggcccccatc tctaaaaatt ttaaattaaa aaaatgttttaacaacaaaa 7500 agcgttctgg gagtgagggg ctggggcctg ggcggcctca ttccatatacctgtgccggg 7560 ttgaggggtt ggagacacgt ttagagaccc ctccactcta ggaatccacctcgagagata 7620 aaggtcccgg ccctagccac acccccagga cacggccaga ggccacctccctaggcgggt 7680 ccctccccac cgccaggttc ctggagcgcg tgcggcgcgt gtgcaggggtagggggccgc 7740 aggcgcgcgg actggagagg cgcgcccctc ccgcgtgttg aaattcaaaagaggcgaacg 7800 gcccccggcg cggcggcgcg gctccggtgg agaggtcaag gcaggggccagtcggaggct 7860 cccggggcgg ggtcgaaccc gcggccaacc tgagcagcag cggaagcttaaagagctcag 7920 gttcccgccc cccggcccta ccatggctac agagcagtgg ttcgaggggtcgctccccct 7980 ggaccctgga gaaacaccgc ctccagacgc cttggaacct gggacgccgccctgcggaga 8040 cccctccagg tcgacgcccc ctggcaggcc tgggaaccca tctgagccggatcctgaaga 8100 tgccgagggg cggctggctg aggcccgggc ctccacgtct tcccccaaacctctggtccc 8160 ccggcctggg ccagcacctc cccgcctatc cctggacact ttgttcagccccatcaccca 8220 acagctgcgc tacctactga agaaggcaga tgatttccag agctacttgctctacaggtg 8280 atgctggaca gggtcccagg tccccatggg taaggagact tggaggggaggcgacaggat 8340 gggtgacaca caccagggtc gcaaaattac aagcgctagg agccagagggagacagtgga 8400 agaagctagc atattagaat ccagtttaag agaatgagga agactgtagaattgcgggta 8460 ggggatggct gctattactg tcgtggcagg gtgggcctgg ggttgtcaagtctctaggac 8520 tttttctccc agtttttaag tgctgtctta cattttgagc cctgtgctggctaaacaaga 8580 cccacctgag ccaaacttgg cctgcaggac atcagtttga gactccaaaggataatgtga 8640 ttcccagacc aggtttccct gtgactctca atttcagtgt ccattggaatttcctaggag 8700 gctgggttgg gtttgtttgc gtgtttgttt ttgagatgga gtctcactctgtcgcccagg 8760 ctggagtgca gtggtgcaat ctcagctcac tgcaacctcc gcctcccggattgaagcaat 8820 tctctgcctc agcctcccga gtagctggga ttacaggcgc ccaccaacatgtgttgcccg 8880 gctaattttt ttcttttctt agtagagaca gagtttcacc atcttggccagactggtctt 8940 gagctcctga cctcatgatc cacccgcctt ggcctcccaa agtgctggaattacagacgt 9000 gagccaccgc gcctacccga ggctgggttt ttttgttttg ttttgttgttatgtgttttt 9060 ttgaaatgga gtcttgctct gtcacctagg ctggagtgca gtggggcgaactcagctcac 9120 tgcaacctcc gcctcccagg ttcgagggat tctcatgagg ctgtttttttttttttaatg 9180 agacagggtc tcgctctgtc acccaagctg gagtgcaagt ggggcagtcatagctcactg 9240 caccctcgaa ctcctggtct caagcaatct tccacctccc ctcctgggtaactgggacta 9300 caggtgccac catgcccagc taattatttt tgtgtagaga tgggttcttgctatgttgcc 9360 taggcttgtc tggaactcct ggcctcaagc aatcctccag cctcagcctcccaaaactct 9420 aggattgcag gcgtgagcca ctgtgcccag accctgcagg aagctctgggtcctaagtgt 9480 tgtgacactc aggtgtcagc actttaacaa gtgttccaaa tgggtttgatgcaggtaaac 9540 cagaaagatg ttcagaaaag acctgaaact gggggctttt ctaatgggtcaaagccaggg 9600 atacaggttg ggattgagta gaatggggaa aactgcgggg tggggaggggttgtgaggga 9660 ttccaggcaa aggccccctt cttccttcag cagagaccaa gtacagaaggagcagctggc 9720 caaggccatg cccaccttct tacagatgtg tgagccctac ttcctgtacctggaggcagc 9780 cgcgagaagc atacccccca tctatggacc cctgcaggag ctggtccgaaagggggtgtg 9840 tggaggtttc ttagacccca cgcccctttc ttctcgcagc tctgagcctgtggggatggt 9900 ggagggggag gcccactcct cgcaggccag ctgatctcac tgtacccccctcttgtatgc 9960 agctgttaga gatctcccaa cagctgaccc tgcgcctgga acagctggtcctcatgtacg 10020 cttcctttgg gttcgtggac ctggaggaga tgaaccccct taggtaaaatggtaggagac 10080 tcagatgggg ggatgaagga gtccaaggcc cagcctcacc cctccattctctcatgtctc 10140 gccagcatct cctgtttctt ttgcgggagg ttctccatca gcctgtcccatgaggtctcc 10200 atcttcagat actgtgcccc aaccgcctac actgccagcc gcttcccccgctacctctat 10260 aagaagatgc gctggcacct ggaagccacc ccagaggccc ctggtcggggacaagattcc 10320 cttgtggatt agtaagtcct cttacccaaa tcaaagtcct cccctttctatgatgaatgc 10380 caatatgacc ctccaaaccg tcaccagcaa agtgaaaagt gagccagggcccgaggcagt 10440 ggctcacgcc tgtaatccca acactttggg aggccgaggc aggaggatcacttgagctca 10500 agagtttgag atcagcctgg gcaagatggc aagaccctgt ctcaacaacaaagaaattcg 10560 ccaggcgtga tggctggcac ctgtagtccc agctacttgg gaggcttaggcaggaggagc 10620 acttgagccc aggaatcaag gctacggtga gctgtgattg tgccactgcactccaccctg 10680 agtggaagca ataatctgtc tcttaaaaaa aaaaaaaagt gaaccaggaaactaaaggct 10740 tttgaaaggc tacctctatt ttcttaaaac ccaccctccc accaaaataaaagttctcat 10800 cttaaaagta ggctggcagg gagaaaaggc cttggagtca cattcctacctgagaacttc 10860 agggcaactt ctgatgagtt cccacctcaa ctccaaaatt aaagccctcaacagaagtag 10920 ctaggaagct gatcacttct aattacagct ccctcccctc ctagctactttctgtgctat 10980 cgagatactt gggaagacac aggccagagt ccagccaatt cgtgcccacagatccagaag 11040 ctgtggtcca tcggccgatg ggtgccccta ggaccagccg aggatgacctttattcatgg 11100 taggagctag ggcaatagca acgtgggcct gggagctgga gggggaggcagaaccccacc 11160 aaagacaatc caccttccca aacactttgc ttcccttagt agtgatagcattttattgtg 11220 ccctgaaaag cacttcatgc agaccccagt aacaacccat ggagatctatgctattggcc 11280 ccatttaaca aagaaaacag ggtgctcaga gaagttgtta cctgcccaaggacacacagc 11340 tagcagagcg aatggacagg tcaggaccag ttattcagcc tctaggagccattactaagt 11400 ctctgatcaa caaggaaaca agtttccccc gggggttttt cccacccgcagctgaaacaa 11460 agcctctttc acctgagcct ctcactcaaa gggagggact cccgaggggcagggggcact 11520 caagtccagg cctgtctatc cctggccccc ccaccccagg attttgtgcccgcaccgctt 11580 ggggactacc agcagctgct gaccatcggc ttcgaggagc ccacgcccacgctggccacc 11640 gacctgctgg tgcagatcct cacgggccag gcaggccagg cccggcctccgagcgcagcc 11700 gggcctgcgg ggtgggcagc gcaggggtct tgaacctggg gaagagggtaggagctggaa 11760 cttgacagtt ccaaactcca gaataggggg caggggaggg gctcactcgttctcgcagtg 11820 cagccgggcc tcgccttcca aagggccagg ccgagctgac ctgtctgcaccgagtccggc 11880 ttggccgtgg ggccctgaat gcggacacgt cagttttgtg ttaaataaaagaaagaaaga 11940 ggtcacaggc tcagcgtccg ctgcgaatgc cgcgcccctc ccccgggggattgccccacc 12000 cactcgcgtg gccttctggg aaatgtagtc ttttgaaaga agcctggaattcgccaatag 12060 gcggacgaga gtttggcgca tgcgcatagg cgcacatgaa gcaaaaagggaagtggtgcc 12120 cgtcaacacc ggaacccaga aaactgcaag tttagggtac cggggaaattcaacgtccac 12180 tggaggaaga gacttaaggc tacgcccact cccatatttt gacccggaagttatttattt 12240 tagcgtagaa gactactttt cccgacgcgc cccaggaaag tgccctcgatcagtttccta 12300 agggcccgag ttagactttt tttttctctt ccagcttttg ggacttgggggccggacagg 12360 tcgtcgtctt tcttggggta tccggggtgc ggacaaggtg ggagagccctacggtatcca 12420 agctt 12425 51 22255 DNA Homo sapiens 51 caacatgcttgggaccagaa gtgtttccaa tttgggattt tctcaaattt taccggttga 60 gcttccccaatctgaaaatc tgaaatccaa catgcacggc tctgaagtct ttcactgagc 120 ctttgggggaaatatttaac atcctaacag ccctaaacca acgctcaatt agcacaacag 180 tttacaatcttctctaccca cagcctgatg cgaggctctg ggactagact atttagccaa 240 cagttcttgcaaaattaact gacttataag taaatagtaa tttcaacacc tcactgctaa 300 tgctgtaacaactctgcaga cctagggagc aagtacggtt tgcagagcac tgggaaggct 360 ctgaagtgacctttgaactg ggcctcaaaa aattttgggt ttggcaaaag tcaaatctct 420 taggcttcaaattccaggca caaggattgt tgggtttgat ttcattatcc agaagcaatg 480 gggatacagaattgtgatct catgtgtagg gaactgtggg ggttttttct actttaaccc 540 cagtgagactttgtagagtg tggggtagag aaaaggctca tgaatatgcc tgaagcctaa 600 ctcagcacctttctgaggaa ctgactgcca aaatggtaat ggagagggga aaatatgacc 660 tactttcacaagttaccttg actgcctcag ggaaacctgc tgtggtagtg tttcttctgg 720 gtgaaagaccaggtaattac ctgggtgctg gtctcagact taccagtttt gaatccctgt 780 tttaaccactcactatcgat atgaccttgg ataagttacc taacctttct cttactgtcc 840 ttttccgtaaaatggggata acagatagta gttatttcta tgagtggtta tgagaaccaa 900 gctattagatagcgggaaag cacacagtaa gcgttcaagg aactgctatt gttattaaaa 960 gcctcctttggaagaaggac attgaggccc agagagagaa cagaacgtcc agccacacag 1020 caaatccgtgatgaagttgg gactggagta tgggtctcct gagtctcagc ccaggactct 1080 atccctcttcccgagtcctc ggagttcccg gatggagtca catttgttca cggccaggga 1140 ggaaggtttgatggaggcct gcaggaaaca acagccaggc gcaaggcttt gggagttgaa 1200 gcatagcttctgcgagatag aaacaaggtt gacatgggca ctcgtgcaga atgacgggct 1260 ccttttggactcccaggact acagtccctt atgcaccttg ggatctgcgg ctagcccctg 1320 cgtaaagagggacgcgtagt cttttccctg ccccgccctg ccggggcgcc cgcctccgag 1380 gccgccctcgcttcgtcctt cccagcaagc tccgcgccgg cgccggctat tgattggctg 1440 aggcgggagcaggcggctgg ccggcagcag ttactcgggg tttccggtgc gaggccagag 1500 gtggggaagccatcggacgt cggcggtgag gtacgtgcag cggcggccgg tgggcgagac 1560 tatttgagagtgtgcgggcc gggatgttct cggcctgtgg ggaaatcacg ccaactcccc 1620 gcgtgggccgggggctgtct ggggatatgc gcatgcgcgg gcgtgcctcg cggcttgagg 1680 gcgcgcggggcgtgggtggc tgcgcgcgcg gggggcgcac gtggggcctg aggggcgggg 1740 gcggtgccgggagtcccgcc acgtcagtct ccggccctga gccaatcccg cgcccggcct 1800 gccgcgagggggccggttgt gccgggaagt ggctccaggg agaagaggcc tcttccctca 1860 cccgctgtgggagctgcgcc ccgaaagcct gccccggcac gtcgggctct cctgacccgc 1920 caagaccagagagccgttgg cgccctccgc ccgggcctgc cggtccgttt attttaagaa 1980 gctttgtgcgcctgctgtgg ggatttctga tccaggctgc gaagaatttc gaagtctgga 2040 aaatagcaactgtgtttgtt tctaaaggat cttctcctga cccagcatcg ctcatcacaa 2100 tgaagaaccaagacaaaaag aacggggctg ccaaacaatc caatccaaaa agcagcccag 2160 gacaaccggaagcaggaccc gagggagccc aggagcggcc cagccaggcg gctcctgcag 2220 tagaagcagaaggtcccggc agcagccagg ctcctcggaa gccggagggt gtgtgccagc 2280 tctgcgttgccagcgggcag ggggaggagc tgtggggtcg gcctcgcttc tggacttaca 2340 ggccgaggccaggttgtccg ggaggaggag atgtagaatg agaggacagt gctgggggcc 2400 gcggtcccccctgcgctctg gcgagttggc ggagctgccc cctctaagca caggaacaga 2460 gttctggagagaagctccga cgggattaag tcaggtggca gccaaacgag gcacccagtc 2520 aggaaatccaggtcccgtta gaaacacctc agccaccagc agctaactgc ccttcctgtt 2580 tgaggcatttctagaatgat ctgaatggca agaaatgggt tttgtggggg ggaaggagat 2640 ggactagaagttgctccgtg ccatccctgt gtgctgatgc tttacatact tttatgatct 2700 aacaaatatgttcgggtggt agtgagaaat agttgtgtca ttttacaagt aaacagactt 2760 aaagaagttaggcaacgatt actataattt cttgatttaa aagatgtttc gaatctaaat 2820 tctgacaggaactagatttg ctgaatgata ctccattctt gcttctcagt ttccataaaa 2880 aaaaaagttaggcaacattt aactcaaact gatgagtttg gctgggcctg aaaaatccca 2940 accagtggtataatcgtctt ctttctcact ctacccctca tcctctcctg ctgtaggggc 3000 tcaagccagaacggctcagt ctggggccct tcgtgatgtc tctgaggagc tgagccgcca 3060 actggaagacatactgagca catactgtgt ggacaataac caggggggcc ccggcgagga 3120 tggggcacagggtgagccgg ctgaacccga agatgcagag aagtcccgga cctatgtggc 3180 aaggaatggggagcctgaac caactccagt agtcaatgga gagaaggaac cctccaaggg 3240 ggatccaaacacagaagaga tccggcagag tgacgaggtc ggagaccgag accatcgaag 3300 gccacaggagaagaaaaaag ccaagggttt gggtgagcag agggcggctc tttgtgaagc 3360 tggtgaggagagggagtttg gacttgacgt tctctgggcc agtctgttct gccaggattc 3420 aaaggaaaacggtacttctc agagcagcaa gtcactctag tctaatcaaa gccagggatg 3480 tgggggccacggcatagaga gatgcaggag ttaccagcac aaagccttct gggttttgga 3540 gcaactggagcttggcatgg gacctgttct ctctttgaga aaatggagac gggaggctag 3600 ggtaggctcctgtgccagcc agtactacct gctgtgtgac cttgggtgtg tcccttctcc 3660 tctctgggtcttagtttata tttctcttta cagtaagaaa attagactag gccagagttg 3720 aaaacccaaatatctgcata agctgggctt ggccatgggg ccacctgaag atggaggctt 3780 tactgcttccctgattagtt gctctcacta gccaactgag agcaggcaaa actacaggct 3840 gggtgcagtcaggctttttt tttttttttt tttttttaaa taaagaaaag ccagaaatct 3900 agagttatgtgagaactcta gattttttca tagttagcag ctaaaatggt aagagccaaa 3960 caaaacccatccgtgggttg gatttggcac acatgcctgc gaattgcagt ctccatgctg 4020 atctcttgggcccttctggg gaggcagagg gaaggctccc tgactcagtc acaggcaatg 4080 gggaataggcagtgacagtc attttacagc agggtatgta tgtttaagag tctaggccgg 4140 ggtgtggtggctcacgcctg taattgcagc actttgggag gccgaggcgg gtggatcacc 4200 tgagggtcaggagttcgaga acagcctggc caacatgatg aaatcccgtc tctactaaaa 4260 atacaaaaattagctggaca tgctggcaca cgcctgtaat cccagctact tgggaggctg 4320 aggcaggagaatggcttgaa cccgggaggc agaggttgca gtgaactgag attgtgccac 4380 tacatccagcctgggtgaca agagtgaaac tctgtctcaa aaaaaaaaaa aaagaatcta 4440 gaatctaagtcgagtgtcat tatatccatg ttttattcct attccctttt ccccttatgt 4500 atcctcttactttaaagagg aactttaaaa aatcttaggg acgactaggc agagtggctc 4560 acacctgtaactccagcact ttgggaggcc aaggcaggca gattatgagg tcaggagttc 4620 gagaccagcctggccaacat ggtgaaaccc cagttctact aaagatacaa aaaatcagcc 4680 gggcgtggtggcacgtgcct ataatcccag atactcggga ggctgaggca ggagaatcac 4740 ttgaacccgtgaggcaaagt tttcagtgag ctgagatcat gccattgcac tccacctggg 4800 tgacagggtgagactccatc tcaaaaaaag aaaaaggaaa aaatcttaac gtcacataca 4860 tggaaagatcatctttttca ccccccaccc ccaactgaga tggagttttg ctcttgtcac 4920 ccaagctggagtgcactggc gcgatctagc tccctgcaag ctccgcctcc cgggttcaca 4980 ccattctccctgcctcagcc tcccgagtag ctgggactac aggctcctgc taccatgccc 5040 ggctaatttttttgtatttt ttttagtaga gacggggttt catctgtgtt agccaggatg 5100 gttttgatctcctgacctcg tgatccgccc gcctcagcct cccaaagtgc tgggattaca 5160 ggcgtaagccactgcacccc gccttttttt tttaattaat taattttttt agacagagtc 5220 tcgctctgtcccaagctgga gtgcagtggc gcgatctggg ctcactgcaa cctccgcctc 5280 ctgggttcacggcgattctc ctgcctcagc ctcccgagta gctgggacta caggctcctg 5340 ctaccatgcccggctaattt ttttgtattt tttttagtag agacggggtt tcactgtgtt 5400 agccaggatggttttgatct cctgacctcg tgatccgccc gcctcagcct cccaaagtcc 5460 gcctcagcctcccaaagtgc tgggattaca ggcgtaagcc actgtaccct gccttttttt 5520 tttaattaattaattttttt agacagagtc tcgctctgtc accaagctgg agtgcagtgg 5580 cgcgatttgggctcactgca acctccgctt cttgggttca agcgattttc ctacctcagc 5640 ctccggagtaactgggacta caggcgcgtg ccaccacacc aagctaattt ttttgtgtat 5700 gtctttagtagagatggggt ttcaccatgt taggatggtc tcgatctctt gacctcgtga 5760 tccgcctgcctcggcctccc aaagtgctgg gattacaggc atgagccacc ttgcctggcc 5820 gaaagtatcttcattttaaa gttcactgtt tggctactct gttgacaaga gtttagtatt 5880 tctcaaggaggctaagatac ctattccttt ttggatccta cctctatcag gagggtgggc 5940 cttccttgcattgaaacagt atgaaaacag tagccctgaa ttcataagtg ggacaccttt 6000 cttctattggtagagcaggc agtttttttc tcctgccaat ggtgcctact aaggagattt 6060 cactagggtacagtcgttca tttgataagc atttgttgag catatcctct gtgatggtac 6120 tatggacagtactggggcta tagtgagggc aggattgagt tggtccttat ggcaaggaag 6180 gcagctaatcaacaagcaaa atataaagta tgatggggag ggctgtcttc agcactcatg 6240 agtgtgagcccaggcctgga ggggacacct ggagaagagg gtgcatgtct ttgctcctgt 6300 gcttttcagggaaggagatc acgttgctga tgcagacatt gaatactctg agtaccccag 6360 aggagaagctggctgctctg tgcaagaagt atgctgaact ggtcagttcc cccctccgcg 6420 ggcaccttccctgcgttggg aaaatcagca tgccacctgg tgtaaggttg ggggtgcaga 6480 gtcaagtaggtggcttaatt cctgttcagc ttttctctga actatctgtt aaatggggaa 6540 tcacttccagccagcctctt cagggctgtg cagcaagagg agaaactgca tattccttga 6600 aagaaatttctcaaagaatg attccaaggt ggtagagccc ttgttcctgg cctgagtcca 6660 agacaccttgtgatcttgat gcttcttcct caaatacaga tgcatagagc cattatcaca 6720 gttaataaaactaacactag tcacttgata ctttttcctt ttactccaga gcagtcttct 6780 tgtcactgcctcctcatatt ccccatgaca ttgactttta acagaaacta gactagctgt 6840 cttgtaggatgcccccttct agctttgtca tctctgtggt atcattttac ttctttacct 6900 cctggtacatgtaagtgaag tagaagttag ctctaaagct tgatccaatt cagcttcaac 6960 tttttgacaagaattcttca taagtacttc atgttccatc acaataaatg caaagcatgc 7020 tcttcccactttgttgtaac attgttcagt gggttggggg tggggcagcc agattcttcc 7080 atcatcaggtcccttgtcag aatttgaact aacagattta tccattgatg gtcacagcct 7140 gtgtatgtatgtatgtatgt atgtatgtat gtatttattt atttatttat tttttgagac 7200 ggggtcttgctctgtcgccc aggctggggt gcagtggcac gatctcggct cgctgcaagc 7260 tccgccttctgggttcatgc cattctcctg cctcagcctc ccgagtagct gggtctacag 7320 gcgcccgccaccatgctagg ctattttttt tttttttttt ttttttagta gagacggggt 7380 ttcaccgtgttagccaggat ggtctcgatc tcttgacctc gtgatccgcc cgcctcggcc 7440 tcccaaagtgctgggattac aggcttgagc caccacgcct ggcctattta tttatttatt 7500 cagagtcagagtctcgctct gtcaccaggc tggagtgcag tggcgcgatc tcggctcatt 7560 gcaacctccacctcccaggt tcaagcgagt ctcctgcctc agcctcccga gtagctggga 7620 ttacaggtgcatgtcaccat gcctggctaa attttgtatg ttttagtaga gacagagttt 7680 cagtatgttggccaggatgg tcttgatctc ttggcctcgt gatccgcccg tctcagcctc 7740 ccaaagtgctgggattacag gtgtgagcca ctgtgcctgg cctctaagta tttattttaa 7800 aattaattcattccacacac atttattaat attttcctgt aaggaacttt actcatcttt 7860 aaaatggggaatgtcatacc tgcctaatga cattcttgta aggattaaat aaaaggtata 7920 aggaagataagcaccctttt ggagtgatcc agccagggga aaattgctga tgcaagagag 7980 gaaatgagttgctagagtgg tgttgtgagt agaggagggg agctgaggcc tgcccaagaa 8040 gggggcttggctgtggtaac cacatggcta ggtctgtgtg actggaggag aggacggggc 8100 aggtggactggtagatgtgc agcttgtgcc cctgattctc tagtttcttc tgtgttttga 8160 gatttgatgagaacgatgaa atagttgtct ggaaggagag gagtgtgaat agcatatgca 8220 ttgtattgggattgctggtc ttcctgaaat tggtggccat gaatttaaag tgagactctt 8280 caagtagggttgttatagta ctggtgtaaa gcaggaaggt gctttactag ggttgcagta 8340 ctactggggaagggccaaga gagttgaggg tgtaagaaat ccaagccagg taatgtagtt 8400 attttaaaggagagtggaag gatggttgag tcaatggatt ggaggtccta tagggtaaga 8460 gactttctgaggatcacaga tactgattgg aatgagctaa aaagataggt gatggtagtc 8520 ctggactgggatgctggaaa ttgagatagt gggtgtgctc tctggtagtg acaaatctag 8580 atctgcgctgtccaagataa attcgtctct agctaattga catgtggcca gtttgaattt 8640 gaacatgctataaatgtaag atacacatca gcttttgaag acttaagcaa aaacaaagaa 8700 tataaaacatctttttgtga gagagtgtct cagtcaccca ggctggagtg cagtggcgtg 8760 atgtcctgcttccaggttca aacgattctc ctgcctcaca gcctcctgga gtaactgaga 8820 ttacaggcgcatgccaccaa actggctact tttttgtatt ttttttttag tagaaacggt 8880 ttcaccatgttggccaggct ggtcttgaac tcctgacctc aagtgatctg cctgcctcag 8940 cctcccaaagtgctgggatt acaggcatga gccaccactc ccggcctcac ttttttacat 9000 tgattccgtgttgaaattgt aatgttttgg atattaggtt aaatacatat attactaaaa 9060 ttaatttcacctgtttttta cttttttagt gcggccagta gaatattttt aattacttat 9120 gtggtttgcattatatttct gttgtacagg cctggatagg gtcatgggag gggaactgag 9180 ctggggaaaggagtgggttt gtggaagagg tgatggactg tgaggccagg gagttagaag 9240 gattatctgttgatactgaa gtggccacaa atgagaaaag taattgtgtt ggggagagcg 9300 ctgatgaacgcagcgctaac gttttgaagg aatgcgaggg agcgatgggg gtctgtctgt 9360 taataggcacaaggtacggt agcaggtggt ctcatcctcg ggcatgagtg tccagcaagt 9420 tggggaaatgcaacagcttg aagtggctct agtggcccag agtcagagct ggaataggaa 9480 ttggcatctgctggctgtgt ggcccctgct tgccctagtg agttaccatt tctctgtccc 9540 tacggtggagcctttggggt tattgtgagt tcatgggagg agcgtgtaag caccggcaca 9600 gcatcagcccatgagagtgc tcctggcctg agagggtaag ggtcagggca gctcaggaga 9660 ccctagacctgcatagtgat ccccccacca ggaaggcccc acaagatgct cacctgccct 9720 ccctatccctgtccccagct ggaggagcac cggaattcac agaagcagat gaagctccta 9780 cagaaaaagcagagccagct ggtgcaagag aaggaccacc tgcgcggtga gcacagcaag 9840 gccgtcctggcccgcagcaa gcttgagagc ctatgccgtg agctgcagcg gcacaaccgc 9900 tccctcaaggtaggcctggg ccccctggaa caggtgactc tggtttcctt gacttccact 9960 taatgtttctttcatgggct ttcctcttaa aaagtagtgc aggctagggc caggcgcagt 10020 ggcacacataagtgattaaa aatcttctgg ccactaaaaa acagaaatta attttagtaa 10080 tatacttaacccaatatcca aaacattaca atttcaacat gaaatcagtg taaaaaagca 10140 aggctgggtgtggtggctca cacctgtaat cccaacactt tgggaggctg aggtggatgg 10200 atcacttgaggccaggagtt tgagaccaac ctggtcaacg cagtgaaacc ccattctact 10260 aaaaatacaaaaattagccg agtgtgctgg caaatgccta taatcccagc tactcaggtg 10320 gctcaggcatgagaattgct tgcacctggg aggctgaggt tgcagtgagc cgagattgca 10380 tcactgcattacagcctggg caacagagtg agactcagtg tccaaaaaaa aaaaaaagta 10440 gtgcaggcttgtggcataga aatacacttt ctcaataatg ccttacgtta agagagtact 10500 gcttgtaatcatttgacatg tattagataa ggtgaaggat aaagtactaa gagaatccat 10560 aatgcactggcgttagtatt tctcaatgaa atgacagtcc cctggtaagc ggaggcctgg 10620 ctctgacaagcagctcttgt cccagacgtt ggtcagtcag gaacctgggt ccttcccatg 10680 ttctgctgcttctatggtga ggtcagtctg tggttacacc aagtttaaat acagcctttt 10740 aactttcttttttatatgta aaatcttaca tgtagttttt agaatgaaat tattatacat 10800 gtaccatttcatatcctgtg cctttttttc actttacata acatttttcc ctatcagtat 10860 gtgtagggctatcttctcat tatatggata tattatatca gtgccctagt taaagcattt 10920 tgggggttgtttacaatttt tcattattac atatagaact atagtgaaaa ttcttgttat 10980 atttatcactggtcagttat atagaactta tctgtaggat aagtcatgga attgaaatgg 11040 ctaggtcacagtatatgcag atttttcatt ttaatagatt ttgctggatt gccttccagt 11100 gagggggcagtgtgccttcc ccatcaaaag tgttgagtgc ctaattctgc acaactttgc 11160 aaaccctgggtgttactaaa ttttaacagc ttggtctctg ggggtacaga ggggacaaat 11220 gcacattaatctgaaatctg gaagaatagg ccttaggaga tccgacttgc ttcagaatgg 11280 cacttagcacttacatgtgt gcatgtgtgc ctgcattttt tcttcctttt tttttttttg 11340 gggacggagtcttgctctgt ggcccatcgc ccaggctgga gtgcagtggc gcgatcatag 11400 ctcaccacaacctccgcctc ccaggttcaa atgactcctc tgcctcagcc tcccaagcag 11460 ctgggaccacaggtgcacac catcacgccg gctaattttt gtattttagt agaaacgggg 11520 tttcaccatattggccaggc tggtctcaaa ctcctgacct cgtgatccgc ccacctcagc 11580 ctcccaaagtgctgggatta caggcgtgag ccaccgcgcc tgccatgtgc ctgcattttt 11640 ctagggggagaatctcactt gatgtcacct gatatacaga ggggcccatt ggaacccgca 11700 ttgcacaacatcctggagtc tggctactcc acgctttggg agcagggagg gctgttggca 11760 gagaccatctgtggactagc tgggggaccc ttgtgaggta gcagtggatg atggctctcg 11820 ggctgacttctttgcccagg aagaaggtgt gcagcgggcc cgggaggagg aggagaagcg 11880 caaggaggtgacctcgcact tccaggtgac actgaatgac attcagctgc agatggaaca 11940 gcacaatgagcgcaactcca agctgcgcca agagaacatg gagctggctg agaggctcaa 12000 gaagctgattgagcagtatg agctgcgcga ggaggtaagg gtatcacgga cagcagtcat 12060 ggcccagaaattgtgaggtt ttgagtgtgt gctaggcact gggacagtac cttttcaggc 12120 ttcatcccattctccctttc ttcctcctcc tcctccttgg gaggagagta atgttattcc 12180 tcatagataaaaaacaggtg tggagaagag actcacttac agccacacag ccccaggtcc 12240 acagtgccttgtcccaaatg actgggccag gcatcttttg gaattagaac tatccacatt 12300 ttagaatggaggtacatgta tggactgtgt gttatatagc accctcagca gggccttggg 12360 gaagccagacacattaatgt atttatgcag tagaacttcc aaatactcac ctacattatg 12420 ggcttacaatgatgcaggtc aagtctggct gccagcttat gacaatttcc attttcagaa 12480 ctttgtagaatttggaattg caggggaggg gtgtacctgt gatcagtgat ggactccaga 12540 gactgtgtccactgattcct tgctgctcct gccactcaaa aggcagaatt tatcaggctg 12600 ggcgtggtggctcatgcctg taatcccaac actttgggag gccaaagcgg gcggatcacc 12660 tgaggtcaggagttcaagac cagcctggcc aacatggtga aaccctgtct ctactaaaaa 12720 tacaaaaaattagccaggtg tggtggtgca cggctgtagt cccagctact caggaggctg 12780 aggcaggagaattgcttgaa cccaggaggc agaggttgca atgagccaag attgtgctac 12840 tgcactctagcctgggtgat ataccgagac tccatctcaa aaaaaaaaaa aaaaaaaagc 12900 aggatgtcactccctttgtc actgcgttgg ctgccacccc aggcacttga atctttggat 12960 cttccctgccagtcacctgg ctgttctggg cgcgttctca tcatgagaag ggagacctgc 13020 agcccccttacagggctggc agaggacctg ctctggatta ggccctttcc tagcccctgg 13080 ggtgtggcagtgggtgagac cgggaagatc tgccctctta ggttcatagg ccaaagtgat 13140 gatcgtgtgtgcaggaccta gagggcgctc ccctgaccca cccctttcct tgccatactt 13200 catcctctgggaacaaagct gcttgtttgg tttgagggga gttggtttgg ttcttatccc 13260 tcagcgctgagacatagagg cttcctgggc cactacagtg agacacgaac ttcaagaatc 13320 tgaatacccccgttttctct ccccgccaag gcaaaaaagg acttagtact acctgtggag 13380 aaggaggtgcaggactacca ggccctgctg ctttgcattt acagccctcc ccagacagac 13440 acaggcaccctcatcatacc caaactggac ttacctgcta ggcaccttcc cttccccatc 13500 caaaaaaatggagttatttt cccttatttc agcaagtcca gttgatttta cctttgaagt 13560 agcacctgagtccttcacct tctctccatc ccttctctct cacctgacac aggtctgcag 13620 cgctcctctagtaggcagga cagccattcc ttggggatgc acatgtctag tctttgccta 13680 gatatggcaagtctttgcca actgagctag gctgttatgt tcttagaggc attgtttttg 13740 cccattcttcccatttacaa gagaatcagg gacacagaag tgagggcttc cagccccata 13800 ggtgatcaatcctggggtca gagatttgag tgtgtttatt gcttgccttc ttgggagcag 13860 attccatccataaaccatgt gcttaccaag gtctgactca ctgggagaga aacgacgtga 13920 ggttggaaagctgaccttcc agagacttgg ggcccatgtt gtgtggtaca catgggagtc 13980 catcatatcagattgagatg gggggctggg caaagtgccc tggtctgtgg ctgtggggct 14040 accctgagaaagggagcgcc tgacaagccg actgctccca ccatctttgt tgcagcatat 14100 cgacaaagtcttcaaacaca aggacctaca acagcagctg gtggatgcca agctccagca 14160 ggcccaggagatgctaaagg aggcagaaga gcggcaccag cgggagaagg attttgtgag 14220 gctcaggccccagggttggg gtgggggtgt gggaggagac aggctgggct ctggctcagc 14280 tcatagccgggttatatggg agaagtctgg ccagaccagg cacagattcc ttgagtacca 14340 gtctgagagcaggaagcctc agtgggtctg gtgcttgtgg ctaaaaacca aacatagccc 14400 ctgggggcttctgacaggat ctggggttct gtcttggaaa tagctcctga aagaggcagt 14460 agagtcccagaggatgtgtg agctgatgaa gcagcaagag acccacctga agcaacaggt 14520 gagagcatataacctgaccc tgtgccttca agtttccctc actgggcccc atcctggggg 14580 tagtgaaatgggaccctcat tctaggactg gctgtgtcct ggctgctatg acgccttggt 14640 tgagcttaggtgggctcaga ggacttcatt tgtagctcag aaatgtattg cttttgagga 14700 ggtaggaacagaagagtttg aaaatcaaca taaaggcaaa ataaaagtca ccctaagtct 14760 cctactttccaggcttagca ttttggatta tatccttcca aatatatagc tttgctttgt 14820 tttaaggaaaaatagtatct caatagaatt actggtcaga gagtcaagga cgggtctgag 14880 tgtgttgaccagagtgcctc ccagagaaac ccagtcttat ctgtgggctg ctttctcccc 14940 acagcttgccctatacacag agaagtttga ggagttccag aacacacttt ccaaaagcag 15000 cgaggtattcaccacattca agcaggagat ggaaaaggta actgtggtcc aggccaggca 15060 tggctgctggggcataagct gcttcattca aaattgttgg gcctgccttc aggaagctcc 15120 catctggggtgtctcaaggg cagggctgtt aggaaggttc acagcctttc ccctcttgag 15180 gcagtatcagtggtatgtat acactccagg ttgtcccagg gaatggggca gtcttttctg 15240 tttgtttggtttttttgggg ggtttgttgt tgttgttgtt gttgttgttg ttgtttgaga 15300 tggagactcacctattgccc aggctggagt gcagtggcat gatctcagct cattgcagcc 15360 tttgccccccgggttcaagt gattctcctg cctcagcctc ctgactagct ggaattacag 15420 gcgcgtgccaccatgcctgg ctaatttttt ctttcttttt tttttgtatt tttagtagag 15480 acggggtttcaccatgttgg ccaggctggt ctcgaactct tggcctcaag tgatctgccc 15540 gccttggcctcccaaagtgc tgggattata ggcgtgagcc accatgcctg gccccttacc 15600 attccttgttattggtggtg gacacctctg acttcctggt ggtgaggtgg cacagagggc 15660 attgactgcatcctgtaatg ccttgcgcct tgggatcaat cattccccac cttggagaca 15720 caggtgcagtccccaccttg gagacacaga ccttggagag gccagctctg accatttcct 15780 tctgtctgtcacataaccta gatgactaag aagatcaaga agctggagaa agaaaccacc 15840 atgtaccggtcccggtggga gagcagcaac aaggccctgc ttgagatggc tgaggaggtg 15900 ggctgtctgtgatctgcagc cagggtgggg gtgtgcactt agcgcatatc aggccctttc 15960 ctgtatgttctacccatcag tgacacagct agcatgaggt agaggtgaga tttgcacaca 16020 atgtccaagtccaaagttaa tgctgttctc tccccatggg aggtggtgag cccagtggta 16080 ggtctccagtgggagtgaag ggagcaaatg gaagaaagga ataaaagagc agaaaaaaac 16140 gggtgccagtgatgtgcctg gtttacatgt aaagcagccc aggtagtttg tgatttcaca 16200 gcttgtaatgtagaagaaag gaactaacga tggagcagca actgcaagcc agaccttgct 16260 gaaagtttttgggttttttt tgtctttttt gctgctgaat gtttttaggt acgttgttca 16320 ttgaaccttctcttgagctc tgaggatggt attagtagtc ctgttttata gatgagacag 16380 gctcaaaagtcaagtccttt gccaaggtca cgtggtagat aaatggagga atacgttatc 16440 tccaagccgtgccccttttc tgcaccatgc tgccccacct gacagcctag tcatggcttc 16500 aactaggactgtttcctaaa gggggccagc tttggactcg gtctgctctc agccttgtta 16560 aagtgtttgccgccaagtgg tgatggtaag tgggaggttg atggggcacg gcactgaagg 16620 tctcatttctttccctagaa aacagtccgg gataaagaac tggagggcct gcaggtaaaa 16680 atccaacggctggagaagct gtgccgggca ctgcagacag agcgcaatga cctgaacaag 16740 agggtacaggacctgagtgc tggtggccag ggctccctca ctgacagtgg ccctgagagg 16800 aggccagaggggcctggggc tcaagcaccc agctccccca gggtcacaga agcgccttgc 16860 tacccaggagcaccgagcac agaagcatca ggccagactg ggcctcaaga gcccacctcc 16920 gccagggcctagagagcctg gtgttgggtc atgctgggaa gggagcggca gcccagccag 16980 gcctggcccataaaaggctc ccatgctgag cagcccattg ctgaagccag gatgttctga 17040 cctggctggcatctggcact tgcaattttg gattttgtgg gtcagtttta cgtacatagg 17100 gcattttgcaaggccttgca aatgcattta tacctgtaag tgtacagtgg gcttgcattg 17160 gggatgggggtgtgtacaga tgaagtcagt ggcttgtctg tgagctgaag agtcttgaga 17220 ggggctgtcatctgtagctg ccatcacagt gagttggcag aagtgacttg agcatttctc 17280 tgtctgatttgaggctcaga cccctccctg cccttcagag ctcaagacaa gtaatacacc 17340 caggtcttgactgcatttgt cttgtgagca gggcttgctt ggtcagctca ggccctccta 17400 gctgctctggaggctccttt gattctctag acctggaaaa ggtgtcccta ggcagagccc 17460 tggcagggcgctcagagctg gggatttgct gcctggaaca agggacctgg agaatgtttt 17520 tgcgtgggatgatgtgctgg tcaggagccc cttgggcatc gcttcccctg ccctttggta 17580 gtgccaggaccaggccaatg atgcttctca gtagccttat cattcacagg tgcctctcta 17640 gcctgcacaaatgattgaca agagatcacc caaaggatta tttctgaagg tgtttttttc 17700 tttatttctttttctttttt tttttttttc tttttctttt ttttttgcac atgacagtgt 17760 ttgtattgaggaccttccaa ggaagaggga tgctgtagca gtggtgcctg ggtgcctggc 17820 ctccagtgtcccacctcctt caccacccca cttggctcct ttgccatctt gatgctgagg 17880 tttcctgtttggtgagatca ggttgtttgt ggtaaaagaa aggaaagggc ttctgatggc 17940 tttgccacaagcttacctgt gggtttcagt cctgagaggc caccaccagt tcccatcagc 18000 actgtctccatgcagcagtt gctgggtccc atgtccagct gcctctttgg cttcatgggt 18060 ttttctgcttcctgccccca cccccacatg tgcaatcctc aagatttgtc ctgattctat 18120 ttcctggcacctccctgcct gtccttgggg attctacttc ttcctgtgtg ggagcccata 18180 gctgttgtctaacaggtaag aaatgaaatt gaactattga ctgggcccca gaaatccata 18240 aaatggctgcagacagttgt ttctgtgtcc tgttctaccc ccactccagt acataactac 18300 tatgtactgtgtagagccat tctatatgct gaatgttctg ctgttgcaaa cttgccaggg 18360 tattagccagtgtttgtgcc aagcagtttt ctgggacaac agaatgactc agaccaagat 18420 ggataggatggttagggctt tgcttcttgc tgtttttctt tgaagctagt tcattgtcct 18480 gcaggtcccttcatcttcca tacctagccc actcttttag cccttacctt aaatctctca 18540 gataagttggttcacaaaga atgttaagta ctgaatcatg tgtgactgag accagagatg 18600 gcaaatgaatggcacaccat ttctccttct cctgccccag ggcaggtacc actgatctgc 18660 atcagagttgcctgctattc tctggtgtat ccttcacatc taggtgccct caagcagctg 18720 tgtgagtgttgagatctctg ccatctctgg ctgagatact gctgtcctgt gaagtgtttc 18780 ccatgacctttttcttcccc tttgaatccc tctgtctgga gtagtccttg cctcttcctg 18840 ctccagtagggccttttccc taccccagcc cctgtgccag gctaagctgg tacaagagct 18900 gccaacctcacagagtgttt gctaggcgag agaggtgcag ggaagaggca gaggtatgca 18960 ccttcccccttgaagagagg ggaaaggcct acagtggccc acataattgc ctgactcaca 19020 cttcagctacctcttaatgc ctgtggaggg actggagctg ctggatccca gtgtggtggt 19080 gtaggaggccacagtgagca ggtggcccca gctgggtttc ccaggtcagg aatgtgggcc 19140 ccaggcaaggtgcagccttt gctcacagct ccatccatgt ctagaccttc aggccagtct 19200 gcagatgaggttccctacct ttttcttctc ttcattgacc aaatcaacca atcactacag 19260 ctgctctgcttctgctttcc aaagtagccc aggtcctggg ccagatgcag gggaggtgcc 19320 tatccatgagtgaaggccag tgtcttcctc acctgggtgg gtcccacact tgtgacctca 19380 gttttaggaccaagatctgt gttggtttct tagattgcta gcttttcctc caggggacca 19440 cagcaggtgaagctcaagag cgcatggctc tgctaatagt aaattgtttt cagggccttg 19500 tccagctgagagcttcatgt ccaccagatt ctgagaggtg tcagcagcac tttttttttt 19560 tatttgttgtttgttttcca tgaggttatc ggaccatggg ctgagctcag gcactttctg 19620 taggagactgttatttctgt aaagatggtt atttaaccct tctcacccca tcacggtggc 19680 cctgagggctgacccggagg ccagtggagc tgcctggtgt ccacggggga gggccaaggc 19740 ctgctgagctgattctccag ctgctgcccc agcctttccg ccttgcacag cacagaggtg 19800 gtcaccccagggacagccag gcacctgctc ctcttgccct tcctggggga agggagctgc 19860 cttctgtccctgtaactgct ttccttatgg cccagcccgg ccactcagac ttgtttgaag 19920 ctgcactggcagcttttttg tctcctttgg gtattcacaa cagccaggga cttgattttg 19980 atgtattttaaaccacatta aataaagagt ctgttgcctt acttgtttct ctcctgacct 20040 gtgtattcctttgtttctgg atctgatcca ttcagcccct tccatcatca ctgacttgtt 20100 caggtctgctgcagagcgcc catggtggtt ccctggtatc ttacatattc cacagtgtct 20160 ttgagcagtcgccacagcct caggatgctg gcatattcac ttgagctgcc tgagtggagc 20220 ccttggcaaagttggcaaga cccttgcctc agagaggatc acacacacac aaaaaagttt 20280 tccctgacctgggggctcac aggctagtga agggaaaagg tacttttagc tatagacagg 20340 tcaatggtgctgagagcaga gaggaggccc ctgccccctt cagcaaggtg agggggtgat 20400 acctggaatggccttctgaa ccacagggca ggtagaagat gaacgtcatt tagtgattaa 20460 atggtacagctgggaagcag gtccatggga ctgggagagg gggtgaggct gggcccagag 20520 tctgggtaccaggttaagga atgtgggcta gatccagagg gcaggggggg caactgaagg 20580 tgtttcaataggaaattgat aggctccagc agtaaggcaa aaggcatgga gccaggcata 20640 ggccatttgaggcccaggtt aagaggggtg gacactcatc actgctattt gggtctgagc 20700 tgtgggtaggctcctatagc cctggcctgc ccaagggaat tcacaggggc ctctaattgt 20760 atgcattccttaaggagagc acattctctg ttcagttttt acacccccca tttacccacc 20820 tcaagcatgggactcctata tgggagacat gctgctggtg gcctcaccca gcaccctgtt 20880 ctctctgggtcctgggttgg tcaggcacaa aggatgatat gtgctgaatg cccaggaaat 20940 ggcagagacaacccacctgc ccttccctcc aggcctccac aaatagatgt gcccacaatg 21000 actgtgacagtcccagcaga gcctctgacc cttctagctg ggtcctgata catgttttcc 21060 atgctggccatgttatttct agtcgcagat cctctggagg gtgtgggggg ggtgccgccc 21120 caactcttggagattccaag caaagcagct ctgagaataa tgaggtttct gaccccccag 21180 tgaagcagctgaggatggga accacagggg tgctccctct gtcagcagca ttaccactgt 21240 ctactctagcagctccggtg gggaaggaga gggatttctg ttgtccccag tctgggcccc 21300 tggttattgaaaaagttcgg aattactctt tacccttgtg gagtgttctg agtgttggaa 21360 gtacccaggaagaagccctg agcaggtgcc ctcaggagca gtgcccatgg ctccccacat 21420 cagccaagaggcccaacccc aggaagccac tcctgcccgg ggatggggaa ggtgggctgg 21480 gtggctgtgtgcactgccct gggccagctc acttgagcct gctgagccgc ctggccaaac 21540 atgagcctctctcctgttgt atcagatgct gttctgggga cctgcgccag gagcctctgc 21600 cagggctttaaatagctgcc cccattgatc tggctgcagg cagcagcagt cacactgggt 21660 cagcctccatcaggtgctca ggtttccctg aggactggag tcaggtgcca gggaatcgcg 21720 tggtctaccttatgacctgg tgctccccac acctgtctcc taggcctggg gggtggggag 21780 gactcctgtcacttcatctg cggcaaaata cagcccccac cacttaccag agaaaactgt 21840 ctggcattgtagagagaggg gttttgccct caaaagactg ttgcttactt tcagtagaat 21900 ggggaatgacactggtatct tccttaaggg ttgttatggg gatgaaatgt atgtaaagtg 21960 ctcaatagggcactggactc actccattga tggctgtctt tgctcgaagt gtcttcctga 22020 tgctgctgctgttgctgctt gtgcttcttc tgtgcttaca ttctctctct ctcactcact 22080 cactctgtctctcctctccc ccgccccacc ccctttctga caaagccacc accattttgt 22140 aaggaactgtagcttctctc tgaaactgcc gggaaaggga aaatcttttt aaaatagaca 22200 tcacacaaccaacagggtcc cctaggttca ggcggggagg tgaggtcgag tgaga 22255 52 7 PRTArtificial Sequence Polycatonic peptide 52 Arg Arg Arg Arg Arg Arg Arg 15 53 7 PRT Artificial Sequence Polycatonic peptide 53 Lys Lys Leu LysLeu Xaa Xaa 1 5

What is claimed is:
 1. An isolated polypeptide that binds to LDL and hasat least 80% sequence identity to the amino acid sequence of SEQ IDNO:7.
 2. The polypeptide of claim 1, wherein the polypeptide has atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:7.3. The polypeptide of claim 1, wherein the polypeptide has at least 95%sequence identity to the amino acid sequence of SEQ ID NO:7.
 4. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:7.5. The polypeptide of claim 4, wherein the polypeptide consists of theamino acid sequence of SEQ ID NO:7.
 6. An isolated polypeptide thatbinds to LDL and has at least 80% sequence identity to the amino acidsequence of SEQ ID NO:43.
 7. The polypeptide of claim 6, wherein thepolypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:43.
 8. The polypeptide of claim 6, wherein thepolypeptide has at least 95% sequence identity to the amino acidsequence of SEQ ID NO:43.
 9. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:43.
 10. The polypeptide of claim 9,wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:43.
 11. An isolated polypeptide comprising a peptide sequence,wherein the peptide sequence binds to LDL and has at least 80% sequenceidentity to the amino acid sequence of SEQ ID NO:7.
 12. The polypeptideof claim 11, wherein the peptide sequence has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:7.
 13. The polypeptideof claim 11, wherein the peptide sequence has at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:7.
 14. An isolatedpolypeptide comprising a peptide sequence, wherein the peptide sequencebinds to LDL and has at least 80% sequence identity to the amino acidsequence of SEQ ID NO:43.
 15. The polypeptide of claim 14, wherein thepeptide sequence has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:43.
 16. The polypeptide of claim 14, wherein thepeptide sequence has at least 95% sequence identity to the amino acidsequence of SEQ ID NO:43.
 17. An isolated polypeptide comprising apeptide sequence that (i) binds to LDL and (ii) is identical to afragment of at least ten amino acid residues of SEQ ID NO:7.
 18. Thepolypeptide of claim 17, wherein the peptide sequence is identical to afragment of at least 20 amino acid residues of SEQ ID NO:7.
 19. Thepolypeptide of claim 18, wherein the peptide sequence is identical to afragment of at least 30 amino acid residues of SEQ ID NO:7.
 20. Thepolypeptide of claim 19, wherein the peptide sequence is identical to afragment of at least 50 amino acid residues of SEQ ID NO:7.
 21. Thepolypeptide of claim 20, wherein the peptide sequence is identical to afragment of at least 100 amino acid residues of SEQ ID NO:7.
 22. Thepolypeptide of claim 17, wherein the peptide sequence comprises SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.
 23. An isolatedpolypeptide comprising a peptide sequence that (i) binds to LDL and (ii)is identical to a fragment of at least ten amino acid residues of SEQ IDNO:43.
 24. The polypeptide of claim 23, wherein the peptide sequence isidentical to a fragment of at least 20 amino acid residues of SEQ IDNO:43.
 25. The polypeptide of claim 24, wherein the peptide sequence isidentical to a fragment of at least 30 amino acid residues of SEQ IDNO:43.
 26. The polypeptide of claim 25, wherein the peptide sequence isidentical to a fragment of at least 50 amino acid residues of SEQ IDNO:43.
 27. The polypeptide of claim 26, wherein the peptide sequence isidentical to a fragment of at least 100 amino acid residues of SEQ IDNO:43.
 28. An isolated polypeptide that binds to LDL and whose sequencediffers by one or more conservative amino acid substitutions from theamino acid sequence of SEQ ID NO:7.
 29. An isolated polypeptide thatbinds to LDL and whose sequence differs by one or more conservativeamino acid substitution from the amino acid sequence of SEQ ID NO:43.30. An isolated polypeptide comprising a peptide sequence, wherein thepeptide sequence binds to LDL and differs by one or more conservativeamino acid substitutions from the amino acid sequence of SEQ ID NO:7.31. An isolated polypeptide comprising a peptide sequence, wherein thepeptide sequence binds to LDL and differs by one or more conservativeamino acid substitutions from the amino acid sequence of SEQ ID NO:43.32. An isolated polypeptide comprising a peptide sequence, wherein thepeptide sequence binds to LDL and differs by one or more conservativeamino acid substitutions from the amino acid sequence of a fragment ofat least 30 consecutive amino acid residues of SEQ ID NO:
 7. 33. Anisolated polypeptide comprising a peptide sequence, wherein the peptidesequence binds to LDL and differs by one or more conservative amino acidsubstitutions from the amino acid sequence of a fragment of at least 30consecutive amino acid residues of SEQ ID NO: 43.